register interest

Professor David Stuart FRS

Research Area: Protein Science and Structural Biology
Technology Exchange: Bioinformatics, Computational biology, Crystallography and Protein interaction
Scientific Themes: Protein Science & Structural Biology
Keywords: Viral Proteins, X Ray crystallographic methods, Viruses, virus-receptor interactions, retroviruses and structure to function of genome
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Viruses are attractive targets for study at the molecular level, since they are sufficiently simple that we may hope to achieve a rather complete understanding of their biology. In practice although their genomes are compact they display astonishing diversity, both in structure and function. Our attempts to relate structure to function have benefited from the developments in X-ray crystallographic methods that have brought very complex structures within reach of description in atomic detail. Our targets range from picornaviruses, small ssRNA viruses, which include a number of important animal and human pathogens, to the larger dsRNA viruses. At both ends of this spectrum (from less than 10,000,000 to about 100,000,000 Daltons) we now have representative atomic structures.

Our efforts are particularly focused on virus-receptor interactions and basic puzzles of virus assembly. Our studies here are highly collaborative, with strong links with a number of virologists (P. Mertens and B. Charleston (Pirbright), D. Rowlands (Leeds), P. Roy (London) as well as numerous groups elsewhere in Europe).

Work on cell-surface molecules is largely performed in collaboration with the group of Prof. E.Y. Jones, whose entry describes many of the projects.

We have a particular interest in studying virus evolution and many of these studies are perfoirmed in collaboration with D. Bamford in Helsinki.

Finally, we are studying a number of viral proteins and enzymes which are potential drug targets and/or illuminate how viruses modulate host responses. For example, the immune modulators of pox viruses.

Name Department Institution Country
Professor Dennis Bamford Institute of Biotechnology and Department of Biosciences University of Helsinki Finland
Dr Bryan Charleston Jenner Institute University of Oxford United Kingdom
Oghbaey S, Sarracini A, Ginn HM, Pare-Labrosse O, Kuo A, Marx A, Epp SW, Sherrell DA, Eger BT, Zhong Y et al. 2016. Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography. Acta Crystallogr D Struct Biol, 72 (Pt 8), pp. 944-955. | Show Abstract | Read more

The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development of novel structure-determination approaches for proteins, and promises visualization of protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to the development of sample-delivery systems that allow these unique sources to be most efficiently exploited for high-throughput serial femtosecond crystallography. Here, the next iteration of a fixed-target crystallography chip designed for rapid and reliable delivery of up to 11 259 protein crystals with high spatial precision is presented. An experimental scheme for predetermining the positions of crystals in the chip by means of in situ spectroscopy using a fiducial system for rapid, precise alignment and registration of the crystal positions is presented. This delivers unprecedented performance in serial crystallography experiments at room temperature under atmospheric pressure, giving a raw hit rate approaching 100% with an effective indexing rate of approximately 50%, increasing the efficiency of beam usage and allowing the method to be applied to systems where the number of crystals is limited.

Ginn HM, Roedig P, Kuo A, Evans G, Sauter NK, Ernst OP, Meents A, Mueller-Werkmeister H, Miller RJ, Stuart DI. 2016. TakeTwo: an indexing algorithm suited to still images with known crystal parameters. Acta Crystallogr D Struct Biol, 72 (Pt 8), pp. 956-965. | Show Abstract | Read more

The indexing methods currently used for serial femtosecond crystallography were originally developed for experiments in which crystals are rotated in the X-ray beam, providing significant three-dimensional information. On the other hand, shots from both X-ray free-electron lasers and serial synchrotron crystallography experiments are still images, in which the few three-dimensional data available arise only from the curvature of the Ewald sphere. Traditional synchrotron crystallography methods are thus less well suited to still image data processing. Here, a new indexing method is presented with the aim of maximizing information use from a still image given the known unit-cell dimensions and space group. Efficacy for cubic, hexagonal and orthorhombic space groups is shown, and for those showing some evidence of diffraction the indexing rate ranged from 90% (hexagonal space group) to 151% (cubic space group). Here, the indexing rate refers to the number of lattices indexed per image.

Stuart DI, Subramaniam S, Abrescia NG. 2016. The democratization of cryo-EM. Nat Methods, 13 (8), pp. 607-608. | Read more

Zhao Y, Ren J, Harlos K, Jones DM, Zeltina A, Bowden TA, Padilla-Parra S, Fry EE, Stuart DI. 2016. Toremifene interacts with and destabilizes the Ebola virus glycoprotein. Nature, 535 (7610), pp. 169-172. | Show Abstract | Read more

Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein–drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.

Ginn HM, Stuart DI. 2016. Recovery of data from perfectly twinned virus crystals revisited. Acta Crystallogr D Struct Biol, 72 (Pt 6), pp. 817-822. | Show Abstract | Read more

Perfect merohedral twinning of crystals is not uncommon and complicates structural analysis. An iterative method for the deconvolution of data from perfectly merohedrally twinned crystals in the presence of noncrystallographic symmetry (NCS) has been reimplemented. It is shown that the method recovers the data effectively using test data, and an independent metric of success, based on special classes of reflections that are unaffected by the twin operator, is now provided. The method was applied to a real problem with fivefold NCS and rather poor-quality diffraction data, and it was found that even in these circumstances the method appears to recover most of the information. The software has been made available in a form that can be applied to other crystal systems.

Ginn HM, Evans G, Sauter NK, Stuart DI. 2016. On the release of cppxfel for processing X-ray free-electron laser images. J Appl Crystallogr, 49 (Pt 3), pp. 1065-1072. | Show Abstract | Read more

As serial femtosecond crystallography expands towards a variety of delivery methods, including chip-based methods, and smaller collected data sets, the requirement to optimize the data analysis to produce maximum structure quality is becoming increasingly pressing. Here cppxfel, a software package primarily written in C++, which showcases several data analysis techniques, is released. This software package presently indexes images using DIALS (diffraction integration for advanced light sources) and performs an initial orientation matrix refinement, followed by post-refinement of individual images against a reference data set. Cppxfel is released with the hope that the unique and useful elements of this package can be repurposed for existing software packages. However, as released, it produces high-quality crystal structures and is therefore likely to be also useful to experienced users of X-ray free-electron laser (XFEL) software who wish to maximize the information extracted from a limited number of XFEL images.

DiMattia MA, Watts NR, Cheng N, Huang R, Heymann JB, Grimes JM, Wingfield PT, Stuart DI, Steven AC. 2016. The Structure of HIV-1 Rev Filaments Suggests a Bilateral Model for Rev-RRE Assembly. Structure, 24 (7), pp. 1068-1080. | Show Abstract | Read more

HIV-1 Rev protein mediates the nuclear export of viral RNA genomes. To do so, Rev oligomerizes cooperatively onto an RNA motif, the Rev response element (RRE), forming a complex that engages with the host nuclear export machinery. To better understand Rev oligomerization, we determined four crystal structures of Rev N-terminal domain dimers, which show that they can pivot about their dyad axis, giving crossing angles of 90° to 140°. In parallel, we performed cryoelectron microscopy of helical Rev filaments. Filaments vary from 11 to 15 nm in width, reflecting variations in dimer crossing angle. These structures contain additional density, indicating that C-terminal domains become partially ordered in the context of filaments. This conformational variability may be exploited in the assembly of RRE/Rev complexes. Our data also revealed a third interface between Revs, which offers an explanation for how the arrangement of Rev subunits adapts to the "A"-shaped architecture of the RRE in export-active complexes.

Zhao Y, Ren J, Harlos K, Stuart DI. 2016. Structure of glycosylated NPC1 luminal domain C reveals insights into NPC2 and Ebola virus interactions. FEBS Lett, 590 (5), pp. 605-612. | Show Abstract | Read more

Niemann-pick type C1 (NPC1) is an endo/lysosomal membrane protein involved in intracellular cholesterol trafficking, and its luminal domain C is an essential endosomal receptor for Ebola and Marburg viruses. We have determined the crystal structure of glycosylated NPC1 luminal domain C and find all seven possible sites are glycosylated. Mapping the disease mutations onto the glycosylated structure reveals a potential binding face for NPC2. Knowledge-based docking of NPC1 onto Ebola viral glycoprotein and sequence analysis of filovirus susceptible and refractory species reveals four critical residues, H418, Q421, F502 and F504, some or all of which are likely responsible for the species-specific susceptibility to the virus infection.

Zhu L, Wang X, Ren J, Kotecha A, Walter TS, Yuan S, Yamashita T, Tuthill TJ, Fry EE, Rao Z, Stuart DI. 2016. Structure of human Aichi virus and implications for receptor binding. Nat Microbiol, 1 (11), pp. 16150. | Show Abstract | Read more

Aichi virus (AiV), an unusual and poorly characterized picornavirus, classified in the genus Kobuvirus, can cause severe gastroenteritis and deaths in children below the age of five years, especially in developing countries(1,2). The seroprevalence of AiV is approximately 60% in children under the age of ten years and reaches 90% later in life(3,4). There is no available vaccine or effective antiviral treatment. Here, we describe the structure of AiV at 3.7 Å. This first high-resolution structure for a kobuvirus is intermediate between those of the enteroviruses and cardioviruses, with a shallow, narrow depression bounded by the prominent VP0 CD loops (linking the C and D strands of the β-barrel), replacing the depression known as the canyon, frequently the site of receptor attachment in enteroviruses. VP0 is not cleaved to form VP2 and VP4, so the 'VP2' β-barrel structure is complemented with a unique extended structure on the inside of the capsid. On the outer surface, a polyproline helix structure, not seen previously in picornaviruses is present at the C terminus of VP1, a position where integrin binding motifs are found in some other picornaviruses. A peptide corresponding to this polyproline motif somewhat attenuates virus infectivity, presumably blocking host-cell attachment. This may guide cellular receptor identification.

Ren J, Nettleship JE, Males A, Stuart DI, Owens RJ. 2016. Crystal structures of penicillin-binding protein 3 in complexes with azlocillin and cefoperazone in both acylated and deacylated forms. FEBS Lett, 590 (2), pp. 288-297. | Show Abstract | Read more

Penicillin-binding protein 3 (PBP3) from Pseudomonas aeruginosa is the molecular target of β-lactam-based antibiotics. Structures of PBP3 in complexes with azlocillin and cefoperazone, which are in clinical use for the treatment of pseudomonad infections, have been determined to 2.0 Å resolution. Together with data from other complexes, these structures identify a common set of residues involved in the binding of β-lactams to PBP3. Comparison of wild-type and an active site mutant (S294A) showed that increased thermal stability of PBP3 following azlocillin binding was entirely due to covalent binding to S294, whereas cefoperazone binding produces some increase in stability without the covalent link. Consistent with this, a third crystal structure was determined in which the hydrolysis product of cefoperazone was noncovalently bound in the active site of PBP3. This is the first structure of a complex between a penicillin-binding protein and cephalosporic acid and may be important in the design of new noncovalent PBP3 inhibitors.

Nave C, Sutton G, Evans G, Owen R, Rau C, Robinson I, Stuart DI. 2016. Imperfection and radiation damage in protein crystals studied with coherent radiation. J Synchrotron Radiat, 23 (1), pp. 228-237. | Show Abstract | Read more

Fringes and speckles occur within diffraction spots when a crystal is illuminated with coherent radiation during X-ray diffraction. The additional information in these features provides insight into the imperfections in the crystal at the sub-micrometre scale. In addition, these features can provide more accurate intensity measurements (e.g. by model-based profile fitting), detwinning (by distinguishing the various components), phasing (by exploiting sampling of the molecular transform) and refinement (by distinguishing regions with different unit-cell parameters). In order to exploit these potential benefits, the features due to coherent diffraction have to be recorded and any change due to radiation damage properly modelled. Initial results from recording coherent diffraction at cryotemperatures from polyhedrin crystals of approximately 2 µm in size are described. These measurements allowed information about the type of crystal imperfections to be obtained at the sub-micrometre level, together with the changes due to radiation damage.

Kelly JT, De Colibus L, Elliott L, Fry EE, Stuart DI, Rowlands DJ, Stonehouse NJ. 2015. Potent antiviral agents fail to elicit genetically-stable resistance mutations in either enterovirus 71 or Coxsackievirus A16. Antiviral Res, 124 pp. 77-82. | Show Abstract | Read more

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the two major causative agents of hand, foot and mouth disease (HFMD), for which there are currently no licenced treatments. Here, the acquisition of resistance towards two novel capsid-binding compounds, NLD and ALD, was studied and compared to the analogous compound GPP3. During serial passage, EV71 rapidly became resistant to each compound and mutations at residues I113 and V123 in VP1 were identified. A mutation at residue 113 was also identified in CVA16 after passage with GPP3. The mutations were associated with reduced thermostability and were rapidly lost in the absence of inhibitors. In silico modelling suggested that the mutations prevented the compounds from binding the VP1 pocket in the capsid. Although both viruses developed resistance to these potent pocket-binding compounds, the acquired mutations were associated with large fitness costs and reverted to WT phenotype and sequence rapidly in the absence of inhibitors. The most effective inhibitor, NLD, had a very large selectivity index, showing interesting pharmacological properties as a novel anti-EV71 agent.

Hengrung N, El Omari K, Serna Martin I, Vreede FT, Cusack S, Rambo RP, Vonrhein C, Bricogne G, Stuart DI, Grimes JM, Fodor E. 2015. Crystal structure of the RNA-dependent RNA polymerase from influenza C virus. Nature, 527 (7576), pp. 114-117. | Show Abstract | Read more

Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome. In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching. Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5' and 3' termini of viral genome segments), showing FluPol in transcription pre-initiation states. Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new 'closed' conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols. The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication.

Ilca SL, Kotecha A, Sun X, Poranen MM, Stuart DI, Huiskonen JT. 2015. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes. Nat Commun, 6 pp. 8843. | Show Abstract | Read more

Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems.

Zhu L, Wang X, Ren J, Porta C, Wenham H, Ekström JO, Panjwani A, Knowles NJ, Kotecha A, Siebert CA et al. 2015. Structure of Ljungan virus provides insight into genome packaging of this picornavirus. Nat Commun, 6 pp. 8316. | Show Abstract | Read more

Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses.

Liu C, Zhao Y, He W, Wang W, Chen Y, Zhang S, Ma Y, Gohda J, Ishida T, Walter TS et al. 2015. A RANKL mutant used as an inter-species vaccine for efficient immunotherapy of osteoporosis. Sci Rep, 5 pp. 14150. | Show Abstract | Read more

Anti-cytokine therapeutic antibodies have been demonstrated to be effective in the treatment of several auto-immune disorders. However, The problems in antibody manufacture and the immunogenicity caused by multiple doses of antibodies inspire people to use auto-cytokine as immunogen to induce anti-cytokine antibodies. Nevertheless, the tolerance for inducing immune response against self-antigen has hindered the wide application of the strategy. To overcome the tolerance, here we proposed a strategy using the inter-species cytokine as immunogen for active immunization (TISCAI) to induce anti-cytokine antibody. As a proof of concept, an inter-species cytokine RANKL was successfully used as immunogen to induce anti-RANKL immune response. Furthermore, to prevent undesirable side-effects, the human RANKL was mutated based on the crystal structure of the complex of human RANKL and its rodent counterpart receptor RANK. We found, the antibodies produced blocked the osteoclast development in vitro and osteoporosis in OVX rat models. The results demonstrated this strategy adopted is very useful for general anti-cytokine immunotherapy for different diseases settings.

Ji X, Axford D, Owen R, Evans G, Ginn HM, Sutton G, Stuart DI. 2015. Polyhedra structures and the evolution of the insect viruses. J Struct Biol, 192 (1), pp. 88-99. | Show Abstract | Read more

Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽ 1.5 Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These structures illustrate the effect of 400 million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule's evolution so that surface residues are almost as conserved as the internal residues.

Ren J, Wang X, Zhu L, Hu Z, Gao Q, Yang P, Li X, Wang J, Shen X, Fry EE et al. 2015. Structures of Coxsackievirus A16 Capsids with Native Antigenicity: Implications for Particle Expansion, Receptor Binding, and Immunogenicity. J Virol, 89 (20), pp. 10500-10511. | Show Abstract | Read more

UNLABELLED: Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the primary causes of the epidemics of hand-foot-and-mouth disease (HFMD) that affect more than a million children in China each year and lead to hundreds of deaths. Although there has been progress with vaccines for EV71, the development of a CVA16 vaccine has proved more challenging, and the EV71 vaccine does not give useful cross-protection, despite the capsid proteins of the two viruses sharing about 80% sequence identity. The structural details of the expanded forms of the capsids, which possess nonnative antigenicity, are now well understood, but high resolution information for the native antigenic form of CVA16 has been missing. Here, we remedy this with high resolution X-ray structures of both mature and natural empty CVA16 particles and also of empty recombinant viruslike particles of CVA16 produced in insect cells, a potential vaccine antigen. All three structures are unexpanded native particles and antigenically identical. The recombinant particles have recruited a lipid moiety to stabilize the native antigenic state that is different from the one used in a natural virus infection. As expected, the mature CVA16 virus is similar to EV71; however, structural and immunogenic comparisons highlight differences that may have implications for vaccine production. IMPORTANCE: Hand-foot-and-mouth disease is a serious public health threat to children in Asian-Pacific countries, resulting in millions of cases. EV71 and CVA16 are the two dominant causative agents of the disease that, while usually mild, can cause severe neurological complications, leading to hundreds of deaths. EV71 vaccines do not provide protection against CVA16. A CVA16 vaccine or bivalent EV71/CVA16 vaccine is therefore urgently needed. We report atomic structures for the mature CVA16 virus, a natural empty particle, and a recombinant CVA16 virus-like particle that does not contain the viral genome. All three particles have similar structures and identical antigenicity. The recombinant particles, produced in insect cells (a system suitable for making vaccine antigen), are stabilized by recruiting from the insect cells a small molecule that is different from that used by the virus in a normal infection. We present structural and immunogenic comparisons with EV71 to facilitate structure-based drug design and vaccine development.

Ginn HM, Brewster AS, Hattne J, Evans G, Wagner A, Grimes JM, Sauter NK, Sutton G, Stuart DI. 2015. A revised partiality model and post-refinement algorithm for X-ray free-electron laser data. Acta Crystallogr D Biol Crystallogr, 71 (Pt 6), pp. 1400-1410. | Show Abstract | Read more

Research towards using X-ray free-electron laser (XFEL) data to solve structures using experimental phasing methods such as sulfur single-wavelength anomalous dispersion (SAD) has been hampered by shortcomings in the diffraction models for X-ray diffraction from FELs. Owing to errors in the orientation matrix and overly simple partiality models, researchers have required large numbers of images to converge to reliable estimates for the structure-factor amplitudes, which may not be feasible for all biological systems. Here, data for cytoplasmic polyhedrosis virus type 17 (CPV17) collected at 1.3 Å wavelength at the Linac Coherent Light Source (LCLS) are revisited. A previously published definition of a partiality model for reflections illuminated by self-amplified spontaneous emission (SASE) pulses is built upon, which defines a fraction between 0 and 1 based on the intersection of a reflection with a spread of Ewald spheres modelled by a super-Gaussian wavelength distribution in the X-ray beam. A method of post-refinement to refine the parameters of this model is suggested. This has generated a merged data set with an overall discrepancy (by calculating the R(split) value) of 3.15% to 1.46 Å resolution from a 7225-image data set. The atomic numbers of C, N and O atoms in the structure are distinguishable in the electron-density map. There are 13 S atoms within the 237 residues of CPV17, excluding the initial disordered methionine. These only possess 0.42 anomalous scattering electrons each at 1.3 Å wavelength, but the 12 that have single predominant positions are easily detectable in the anomalous difference Fourier map. It is hoped that these improvements will lead towards XFEL experimental phase determination and structure determination by sulfur SAD and will generally increase the utility of the method for difficult cases.

Materlik G, Rayment T, Stuart DI. 2015. Diamond Light Source: status and perspectives. Philos Trans A Math Phys Eng Sci, 373 (2036), pp. 20130161-20130161. | Show Abstract | Read more

Diamond Light Source, a third-generation synchrotron radiation (SR) facility in the UK, celebrated its 10th anniversary in 2012. A private limited company was set up in April 2002 to plan, construct and operate the new user-oriented SR facility, called in brief Diamond. It succeeded the Synchrotron Radiation Source in Daresbury, a second-generation synchrotron that opened in 1980 as the world's first dedicated X-ray-providing facility, closing finally in 2008, by which time Diamond's accelerators and first beamlines were operating and user experiments were under way. This theme issue of Philosophical Transactions of the Royal Society A gives some examples of the rich diversity of research done in the initial five years, with some glimpses of activity up to 2014. Speakers at the 10 year anniversary symposium were drawn from a small number of major thematic areas and each theme was elaborated by a few speakers whose contributions were placed into a broader context by a leading member of the UK academic community in the role of rapporteur. This introduction gives a summary of the design choices and strategic planning of Diamond as a coherent user facility, a snapshot of its present status and some consideration of future perspectives.

Altun M, Walter TS, Kramer HB, Herr P, Iphöfer A, Boström J, David Y, Komsany A, Ternette N, Navon A et al. 2015. The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages. PLoS One, 10 (1), pp. e0115344. | Show Abstract | Read more

Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.

De Colibus L, Wang X, Tijsma A, Neyts J, Spyrou JA, Ren J, Grimes JM, Puerstinger G, Leyssen P, Fry EE et al. 2015. Structure Elucidation of Coxsackievirus A16 in Complex with GPP3 Informs a Systematic Review of Highly Potent Capsid Binders to Enteroviruses. PLoS Pathog, 11 (10), pp. e1005165. | Show Abstract | Read more

The replication of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), which are the major cause of hand, foot and mouth disease (HFMD) in children, can be inhibited by the capsid binder GPP3. Here, we present the crystal structure of CVA16 in complex with GPP3, which clarifies the role of the key residues involved in interactions with the inhibitor. Based on this model, in silico docking was performed to investigate the interactions with the two next-generation capsid binders NLD and ALD, which we show to be potent inhibitors of a panel of enteroviruses with potentially interesting pharmacological properties. A meta-analysis was performed using the available structural information to obtain a deeper insight into those structural features required for capsid binders to interact effectively and also those that confer broad-spectrum anti-enterovirus activity.

Kotecha A, Seago J, Scott K, Burman A, Loureiro S, Ren J, Porta C, Ginn HM, Jackson T, Perez-Martin E et al. 2015. Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design. Nat Struct Mol Biol, 22 (10), pp. 788-794. | Show Abstract | Read more

Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

Roedig P, Vartiainen I, Duman R, Panneerselvam S, Stübe N, Lorbeer O, Warmer M, Sutton G, Stuart DI, Weckert E et al. 2015. A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering. Sci Rep, 5 pp. 10451. | Show Abstract | Read more

At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller.

Peyret H, Gehin A, Thuenemann EC, Blond D, El Turabi A, Beales L, Clarke D, Gilbert RJ, Fry EE, Stuart DI et al. 2015. Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins. PLoS One, 10 (4), pp. e0120751. | Show Abstract | Read more

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.

Ginn HM, Messerschmidt M, Ji X, Zhang H, Axford D, Gildea RJ, Winter G, Brewster AS, Hattne J, Wagner A et al. 2015. Structure of CPV17 polyhedrin determined by the improved analysis of serial femtosecond crystallographic data. Nat Commun, 6 pp. 6435. | Show Abstract | Read more

The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We use a new physical model for XFEL diffraction, which better estimates the experimental signal, delivering a high-resolution XFEL structure (1.75 Å), using fewer crystals than previously required for this resolution. The crystal lattice and protein core are conserved compared with a polyhedrin with less than 10% sequence identity. We explain how the conserved biological phenotype, the crystal lattice, is maintained in the face of extreme environmental challenge and massive evolutionary divergence. Our improved methods should open up more challenging biological samples to XFEL analysis.

Saibil HR, Grünewald K, Stuart DI. 2015. A national facility for biological cryo-electron microscopy. Acta Crystallogr D Biol Crystallogr, 71 (Pt 1), pp. 127-135. | Show Abstract | Read more

Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided of the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback.

Wang X, Ren J, Gao Q, Hu Z, Sun Y, Li X, Rowlands DJ, Yin W, Wang J, Stuart DI et al. 2015. Hepatitis A virus and the origins of picornaviruses. Nature, 517 (7532), pp. 85-88. | Show Abstract | Read more

Hepatitis A virus (HAV) remains enigmatic, despite 1.4 million cases worldwide annually. It differs radically from other picornaviruses, existing in an enveloped form and being unusually stable, both genetically and physically, but has proved difficult to study. Here we report high-resolution X-ray structures for the mature virus and the empty particle. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, whereas the empty particle harbours only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions that might correspond to receptor-binding sites. Peptide scanning data extend the previously reported VP3 antigenic site, while structure-based predictions suggest further epitopes. HAV contains no pocket factor and can withstand remarkably high temperature and low pH, and empty particles are even more robust than full particles. The virus probably uncoats via a novel mechanism, being assembled differently to other picornaviruses. It utilizes a VP2 'domain swap' characteristic of insect picorna-like viruses, and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between 'modern' picornaviruses and the more 'primitive' precursor insect viruses; for instance, HAV retains the ability to move from cell-to-cell by transcytosis.

Gildea RJ, Waterman DG, Parkhurst JM, Axford D, Sutton G, Stuart DI, Sauter NK, Evans G, Winter G. 2014. New methods for indexing multi-lattice diffraction data. Acta Crystallogr D Biol Crystallogr, 70 (Pt 10), pp. 2652-2666. | Show Abstract | Read more

A new indexing method is presented which is capable of indexing multiple crystal lattices from narrow wedges of diffraction data. The method takes advantage of a simplification of Fourier transform-based methods that is applicable when the unit-cell dimensions are known a priori. The efficacy of this method is demonstrated with both semi-synthetic multi-lattice data and real multi-lattice data recorded from crystals of ∼1 µm in size, where it is shown that up to six lattices can be successfully indexed and subsequently integrated from a 1° wedge of data. Analysis is presented which shows that improvements in data-quality indicators can be obtained through accurate identification and rejection of overlapping reflections prior to scaling.

Ginn HM, Mostefaoui GK, Levik KE, Grimes JM, Walsh MA, Ashton AW, Stuart DI. 2014. SynchLink: an iOS app for ISPyB. J Appl Crystallogr, 47 (Pt 5), pp. 1781-1783. | Show Abstract | Read more

The macromolecular crystallography (MX) user experience at synchrotron radiation facilities continues to evolve, with the impact of developments in X-ray detectors, computer hardware and automation methods making it possible for complete data sets to be collected on timescales of tens of seconds. Data can be reduced in a couple of minutes and in favourable cases structures solved and refined shortly after. The information-rich database ISPyB, automatically populated by data acquisition software, data processing and structure solution pipelines at the Diamond Light Source beamlines, allows users to automatically track MX experiments in real time. In order to improve the synchrotron users' experience, efficient access to the data contained in ISPyB is now provided via an iOS 6.0+ app for iPhones and iPads. This provides users, both local and remote, with a succinct summary of data collection, visualization of diffraction images and crystals, and key metrics for data quality in real time.

El Omari K, Iourin O, Kadlec J, Fearn R, Hall DR, Harlos K, Grimes JM, Stuart DI. 2014. Pushing the limits of sulfur SAD phasing: de novo structure solution of the N-terminal domain of the ectodomain of HCV E1. Acta Crystallogr D Biol Crystallogr, 70 (Pt 8), pp. 2197-2203. | Show Abstract | Read more

Single-wavelength anomalous dispersion of S atoms (S-SAD) is an elegant phasing method to determine crystal structures that does not require heavy-atom incorporation or selenomethionine derivatization. Nevertheless, this technique has been limited by the paucity of the signal at the usual X-ray wavelengths, requiring very accurate measurement of the anomalous differences. Here, the data collection and structure solution of the N-terminal domain of the ectodomain of HCV E1 from crystals that diffracted very weakly is reported. By combining the data from 32 crystals, it was possible to solve the sulfur substructure and calculate initial maps at 7 Å resolution, and after density modication and phase extension using a higher resolution native data set to 3.5 Å resolution model building was achievable.

Mönttinen HA, Ravantti JJ, Stuart DI, Poranen MM. 2014. Automated structural comparisons clarify the phylogeny of the right-hand-shaped polymerases. Mol Biol Evol, 31 (10), pp. 2741-2752. | Show Abstract | Read more

Polymerases are essential for life, being responsible for replication, transcription, and the repair of nucleic acid molecules. Those that share a right-hand-shaped fold and catalytic site structurally similar to the DNA polymerase I of Escherichia coli may catalyze RNA- or DNA-dependent RNA polymerization, reverse transcription, or DNA replication in eukarya, archaea, bacteria, and their viruses. We have applied novel computational methods for structure-based clustering and phylogenetic analyses of this functionally diverse polymerase superfamily, which currently comprises six families. We identified a structural core common to all right-handed polymerases, composed of 57 amino acid residues, harboring two positionally and chemically conserved residues, the catalytic aspartates. The structural conservation within each of the six families is considerable, for example, the structural core shared by family Y DNA polymerases covers over 90% of the polymerase domain of the Sulfolobus solfataricus Dpo4. Our phylogenetic analyses propose an early separation of RNA-dependent polymerases that use primers from those that are primer-independent. Furthermore, the exchange of polymerase genes between viruses and their hosts is evident. Because of this horizontal gene transfer, the phylogeny of polymerases does not always reflect the evolutionary history of the corresponding organisms.

Owen RL, Paterson N, Axford D, Aishima J, Schulze-Briese C, Ren J, Fry EE, Stuart DI, Evans G. 2014. Exploiting fast detectors to enter a new dimension in room-temperature crystallography. Acta Crystallogr D Biol Crystallogr, 70 (Pt 5), pp. 1248-1256. | Show Abstract | Read more

A departure from a linear or an exponential intensity decay in the diffracting power of protein crystals as a function of absorbed dose is reported. The observation of a lag phase raises the possibility of collecting significantly more data from crystals held at room temperature before an intolerable intensity decay is reached. A simple model accounting for the form of the intensity decay is reintroduced and is applied for the first time to high frame-rate room-temperature data collection.

Axford D, Ji X, Stuart DI, Sutton G. 2014. In cellulo structure determination of a novel cypovirus polyhedrin. Acta Crystallogr D Biol Crystallogr, 70 (Pt 5), pp. 1435-1441. | Show Abstract | Read more

This work demonstrates that with the use of a microfocus synchrotron beam the structure of a novel viral polyhedrin could be successfully determined from microcrystals within cells, removing the preparatory step of sample isolation and maintaining a favourable biological environment. The data obtained are of high quality, comparable to that obtained from isolated crystals, and enabled a facile structure determination. A small but significant difference is observed between the unit-cell parameters and the mosaic spread of in cellulo and isolated crystals, suggesting that even these robust crystals are adversely affected by removal from the cell.

De Colibus L, Wang X, Spyrou JA, Kelly J, Ren J, Grimes J, Puerstinger G, Stonehouse N, Walter TS, Hu Z et al. 2014. More-powerful virus inhibitors from structure-based analysis of HEV71 capsid-binding molecules. Nat Struct Mol Biol, 21 (3), pp. 282-288. | Show Abstract | Read more

Enterovirus 71 (HEV71) epidemics in children and infants result mainly in mild symptoms; however, especially in the Asia-Pacific region, infection can be fatal. At present, no therapies are available. We have used structural analysis of the complete virus to guide the design of HEV71 inhibitors. Analysis of complexes with four 3-(4-pyridyl)-2-imidazolidinone derivatives with varying anti-HEV71 activities pinpointed key structure-activity correlates. We then identified additional potentially beneficial substitutions, developed methods to reliably triage compounds by quantum mechanics-enhanced ligand docking and synthesized two candidates. Structural analysis and in vitro assays confirmed the predicted binding modes and their ability to block viral infection. One ligand (with IC50 of 25 pM) is an order of magnitude more potent than the best previously reported inhibitor and is also more soluble. Our approach may be useful in the design of effective drugs for enterovirus infections.

El Omari K, Iourin O, Kadlec J, Sutton G, Harlos K, Grimes JM, Stuart DI. 2014. Unexpected structure for the N-terminal domain of hepatitis C virus envelope glycoprotein E1. Nat Commun, 5 pp. 4874. | Show Abstract | Read more

Hepatitis C virus (HCV) infection remains a major health problem worldwide. HCV entry into host cells and membrane fusion are achieved by two envelope glycoproteins, E1 and E2. We report here the 3.5-Å resolution crystal structure of the N-terminal domain of the HCV E1 ectodomain, which reveals a complex network of covalently linked intertwined homodimers that do not harbour the expected truncated class II fusion protein fold.

Zhao Y, Ren J, Padilla-Parra S, Fry EE, Stuart DI. 2014. Lysosome sorting of β-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor. Nat Commun, 5 pp. 4321. | Show Abstract | Read more

The integral membrane protein LIMP-2 has been a paradigm for mannose 6-phosphate receptor (MPR) independent lysosomal targeting, binding to β-glucocerebrosidase (β-GCase) and directing it to the lysosome, before dissociating in the late-endosomal/lysosomal compartments. Here we report structural results illuminating how LIMP-2 binds and releases β-GCase according to changes in pH, via a histidine trigger, and suggesting that LIMP-2 localizes the ceramide portion of the substrate adjacent to the β-GCase catalytic site. Remarkably, we find that LIMP-2 bears P-Man9GlcNAc2 covalently attached to residue N325, and that it binds MPR, via mannose 6-phosphate, with a similar affinity to that observed between LIMP-2 and β-GCase. The binding sites for β-GCase and the MPR are functionally separate, so that a stable ternary complex can be formed. By fluorescence lifetime imaging microscopy, we also demonstrate that LIMP-2 interacts with MPR in living cells. These results revise the accepted view of LIMP-2-β-GCase lysosomal targeting.

Stuart DI, Abrescia NG. 2013. From lows to highs: using low-resolution models to phase X-ray data. Acta Crystallogr D Biol Crystallogr, 69 (Pt 11), pp. 2257-2265. | Show Abstract | Read more

The study of virus structures has contributed to methodological advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ~13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.

El Omari K, Meier C, Kainov D, Sutton G, Grimes JM, Poranen MM, Bamford DH, Tuma R, Stuart DI, Mancini EJ. 2013. Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution. Nucleic Acids Res, 41 (20), pp. 9396-9410. | Show Abstract | Read more

Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, 12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from 6, 8 and 13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in 8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in 12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the 12 enzyme.

van Berkel SS, Nettleship JE, Leung IK, Brem J, Choi H, Stuart DI, Claridge TD, McDonough MA, Owens RJ, Ren J, Schofield CJ. 2013. Binding of (5S)-penicilloic acid to penicillin binding protein 3. ACS Chem Biol, 8 (10), pp. 2112-2116. | Show Abstract | Read more

β-Lactam antibiotics react with penicillin binding proteins (PBPs) to form relatively stable acyl-enzyme complexes. We describe structures derived from the reaction of piperacillin with PBP3 (Pseudomonas aeruginosa) including not only the anticipated acyl-enzyme complex but also an unprecedented complex with (5S)-penicilloic acid, which was formed by C-5 epimerization of the nascent (5R)-penicilloic acid product. Formation of the complex was confirmed by solution studies, including NMR. Together, these results will be useful in the design of new PBP inhibitors and raise the possibility that noncovalent PBP inhibition by penicilloic acids may be of clinical relevance.

Nettleship JE, Ren J, Scott DJ, Rahman N, Hatherley D, Zhao Y, Stuart DI, Barclay AN, Owens RJ. 2013. Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment. BMC Struct Biol, 13 (1), pp. 13. | Show Abstract | Read more

BACKGROUND: Signal Regulatory Protein γ (SIRPγ) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRPγ is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRPγ. RESULTS: We obtained crystals of SIRPγ by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRPα and SIRPβ. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRPγ and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRPγ and SIRPα but not SIRPβ. Comparison of the three domain structures of SIRPγ and SIRPα showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRPγ in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRPγ were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRPγ: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd = 1.2 +/- 0.3 μM). CONCLUSION: The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRPγ stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution.

Ravantti J, Bamford D, Stuart DI. 2013. Automatic comparison and classification of protein structures Journal of Structural Biology, 183 (1), pp. 47-56. | Show Abstract | Read more

The classification and alignment of multiple three-dimensional protein structures is a powerful way to detect similarities that cannot be discovered from the sequences alone and can help to infer phylogeny. However, the alignment process remains problematic for divergent structures. We have devised a fully automatic pipeline, HSF, drawing its inspiration from well-known structural alignment methods, which given a list of structures not only aligns all pairs but also classifies them fully. We demonstrate proof of principle for the new method by aligning the currently available set of highly diverged virus coat protein structures containing double β-barrels, as well as validating the method with established test sets for multiple structural alignments. The results for the virus proteins are inline with previous observations based on biochemical, genetic and structural studies but go further, since by providing coherent alignments between sets of molecules with marked structural distortion, they facilitate the marshaling of arguments for or against homology. The classification results can therefore be readily interpreted in terms of phylogeny. © 2013 Elsevier Inc.

Ren J, Wang X, Hu Z, Gao Q, Sun Y, Li X, Porta C, Walter TS, Gilbert RJ, Zhao Y et al. 2013. Picornavirus uncoating intermediate captured in atomic detail. Nat Commun, 4 pp. 1929. | Show Abstract | Read more

It remains largely mysterious how the genomes of non-enveloped eukaryotic viruses are transferred across a membrane into the host cell. Picornaviruses are simple models for such viruses, and initiate this uncoating process through particle expansion, which reveals channels through which internal capsid proteins and the viral genome presumably exit the particle, although this has not been clearly seen until now. Here we present the atomic structure of an uncoating intermediate for the major human picornavirus pathogen CAV16, which reveals VP1 partly extruded from the capsid, poised to embed in the host membrane. Together with previous low-resolution results, we are able to propose a detailed hypothesis for the ordered egress of the internal proteins, using two distinct sets of channels through the capsid, and suggest a structural link to the condensed RNA within the particle, which may be involved in triggering RNA release.

Rissanen I, Grimes JM, Pawlowski A, Mäntynen S, Harlos K, Bamford JK, Stuart DI. 2013. Bacteriophage P23-77 capsid protein structures reveal the archetype of an ancient branch from a major virus lineage. Structure, 21 (5), pp. 718-726. | Show Abstract | Read more

It has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. However, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. Bacteriophage P23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. We have solved multiple crystal structures of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. They fit the 14 Å resolution cryo-electron microscopy reconstruction of the entire virus exquisitely well, allowing us to propose a model for both the capsid architecture and viral assembly, quite different from previously published models. The structures of the capsid proteins and their mode of association to form the viral capsid suggest that the P23-77-like and adeno-PRD1 lineages of viruses share an extremely ancient common ancestor.

Porta C, Kotecha A, Burman A, Jackson T, Ren J, Loureiro S, Jones IM, Fry EE, Stuart DI, Charleston B. 2013. Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen. PLoS Pathog, 9 (3), pp. e1003255. | Show Abstract | Read more

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.

Bahar MW, Sarin LP, Graham SC, Pang J, Bamford DH, Stuart DI, Grimes JM. 2013. Structure of a VP1-VP3 complex suggests how birnaviruses package the VP1 polymerase. J Virol, 87 (6), pp. 3229-3236. | Show Abstract | Read more

Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae, infects young salmon, with a severe impact on the commercial sea farming industry. Of the five mature proteins encoded by the IPNV genome, the multifunctional VP3 has an essential role in morphogenesis; interacting with the capsid protein VP2, the viral double-stranded RNA (dsRNA) genome and the RNA-dependent RNA polymerase VP1. Here we investigate one of these VP3 functions and present the crystal structure of the C-terminal 12 residues of VP3 bound to the VP1 polymerase. This interaction, visualized for the first time, reveals the precise molecular determinants used by VP3 to bind the polymerase. Competition binding studies confirm that this region of VP3 is necessary and sufficient for VP1 binding, while biochemical experiments show that VP3 attachment has no effect on polymerase activity. These results indicate how VP3 recruits the polymerase into birnavirus capsids during morphogenesis.

El Omari K, Sutton G, Ravantti JJ, Zhang H, Walter TS, Grimes JM, Bamford DH, Stuart DI, Mancini EJ. 2013. Plate tectonics of virus shell assembly and reorganization in phage φ8, a distant relative of mammalian reoviruses. Structure, 21 (8), pp. 1384-1395. | Show Abstract | Read more

The hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity.

Ravantti J, Bamford D, Stuart DI. 2013. Automatic comparison and classification of protein structures. J Struct Biol, 183 (1), pp. 47-56. | Show Abstract | Read more

The classification and alignment of multiple three-dimensional protein structures is a powerful way to detect similarities that cannot be discovered from the sequences alone and can help to infer phylogeny. However, the alignment process remains problematic for divergent structures. We have devised a fully automatic pipeline, HSF, drawing its inspiration from well-known structural alignment methods, which given a list of structures not only aligns all pairs but also classifies them fully. We demonstrate proof of principle for the new method by aligning the currently available set of highly diverged virus coat protein structures containing double β-barrels, as well as validating the method with established test sets for multiple structural alignments. The results for the virus proteins are inline with previous observations based on biochemical, genetic and structural studies but go further, since by providing coherent alignments between sets of molecules with marked structural distortion, they facilitate the marshaling of arguments for or against homology. The classification results can therefore be readily interpreted in terms of phylogeny.

Borley DW, Mahapatra M, Paton DJ, Esnouf RM, Stuart DI, Fry EE. 2013. Evaluation and use of in-silico structure-based epitope prediction with foot-and-mouth disease virus. PLoS One, 8 (5), pp. e61122. | Show Abstract | Read more

Understanding virus antigenicity is of fundamental importance for the development of better, more cross-reactive vaccines. However, as far as we are aware, no systematic work has yet been conducted using the 3D structure of a virus to identify novel epitopes. Therefore we have extended several existing structural prediction algorithms to build a method for identifying epitopes on the appropriate outer surface of intact virus capsids (which are structurally different from globular proteins in both shape and arrangement of multiple repeated elements) and applied it here as a proof of principle concept to the capsid of foot-and-mouth disease virus (FMDV). We have analysed how reliably several freely available structure-based B cell epitope prediction programs can identify already known viral epitopes of FMDV in the context of the viral capsid. To do this we constructed a simple objective metric to measure the sensitivity and discrimination of such algorithms. After optimising the parameters for five methods using an independent training set we used this measure to evaluate the methods. Individually any one algorithm performed rather poorly (three performing better than the other two) suggesting that there may be value in developing virus-specific software. Taking a very conservative approach requiring a consensus between all three top methods predicts a number of previously described antigenic residues as potential epitopes on more than one serotype of FMDV, consistent with experimental results. The consensus results identified novel residues as potential epitopes on more than one serotype. These include residues 190-192 of VP2 (not previously determined to be antigenic), residues 69-71 and 193-197 of VP3 spanning the pentamer-pentamer interface, and another region incorporating residues 83, 84 and 169-174 of VP1 (all only previously experimentally defined on serotype A). The computer programs needed to create a semi-automated procedure for carrying out this epitope prediction method are presented.

Burkhardt A, Wagner A, Warmer M, Reimer R, Hohenberg H, Ren J, Fry EE, Stuart DI, Meents A. 2013. Structure determination from a single high-pressure-frozen virus crystal. Acta Crystallogr D Biol Crystallogr, 69 (Pt 2), pp. 308-312. | Show Abstract | Read more

Successful cryogenic X-ray structure determination from a single high-pressure-frozen bovine enterovirus 2 crystal is reported. The presented high-pressure-freezing procedure is based on a commercially available device and allows the cryocooling of macromolecular crystals directly in their mother liquor without the time- and crystal-consuming search for optimal cryoconditions. The method is generally applicable and will allow cryogenic data collection from all types of macromolecular crystals.

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Porta C, Xu X, Loureiro S, Paramasivam S, Ren J, Al-Khalil T, Burman A, Jackson T, Belsham GJ, Curry S et al. 2013. Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity Journal of Virological Methods, 187 (2), pp. 406-412. | Show Abstract | Read more

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine. © 2012 Elsevier B.V.

Iourin O, Harlos K, El Omari K, Lu W, Kadlec J, Iqbal M, Meier C, Palmer A, Jones I, Thomas C et al. 2013. Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus. Acta Crystallogr Sect F Struct Biol Cryst Commun, 69 (Pt 1), pp. 35-38. | Show Abstract | Read more

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

El Omari K, Iourin O, Harlos K, Grimes JM, Stuart DI. 2013. Structure of a pestivirus envelope glycoprotein E2 clarifies its role in cell entry. Cell Rep, 3 (1), pp. 30-35. | Show Abstract | Read more

Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.

DiMattia MA, Watts NR, Stahl SJ, Grimes JM, Steven AC, Stuart DI, Wingfield PT. 2013. Antigenic switching of hepatitis B virus by alternative dimerization of the capsid protein. Structure, 21 (1), pp. 133-142. | Show Abstract | Read more

Chronic hepatitis B virus (HBV) infection afflicts millions worldwide with cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a nonparticulate variant of the protein (core antigen, HBcAg) that forms the building-blocks of capsids. HBeAg is not required for virion production, but is implicated in establishing immune tolerance and chronic infection. Here, we report the crystal structure of HBeAg, which clarifies how the short N-terminal propeptide of HBeAg induces a radically altered mode of dimerization relative to HBcAg (∼140° rotation), locked into place through formation of intramolecular disulfide bridges. This structural switch precludes capsid assembly and engenders a distinct antigenic repertoire, explaining why the two antigens are cross-reactive at the T cell level (through sequence identity) but not at the B cell level (through conformation). The structure offers insight into how HBeAg may establish immune tolerance for HBcAg while evading its robust immunogenicity.

Barford D, Stuart DI. 2012. Louise N. Johnson 1940-2012. Nat Struct Mol Biol, 19 (12), pp. 1216-1217. | Read more

Wilson KS, Stuart DI. 2012. Dame Louise Napier Johnson (1940-2012) Obituary ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 68 pp. 1415-1416. | Read more

Wilson KS, Stuart DI. 2012. Dame Louise Napier Johnson (1940-2012). J Synchrotron Radiat, 19 (Pt 6), pp. 1061-1063. | Read more

Bowden TA, Baruah K, Coles CH, Harvey DJ, Yu X, Song BD, Stuart DI, Aricescu AR, Scanlan CN, Jones EY, Crispin M. 2012. Chemical and structural analysis of an antibody folding intermediate trapped during glycan biosynthesis. J Am Chem Soc, 134 (42), pp. 17554-17563. | Show Abstract | Read more

Human IgG Fc glycosylation modulates immunological effector functions such as antibody-dependent cellular cytotoxicity and phagocytosis. Engineering of Fc glycans therefore enables fine-tuning of the therapeutic properties of monoclonal antibodies. The N-linked glycans of Fc are typically complex-type, forming a network of noncovalent interactions along the protein surface of the Cγ2 domain. Here, we manipulate the mammalian glycan-processing pathway to trap IgG1 Fc at sequential stages of maturation, from oligomannose- to hybrid- to complex-type glycans, and show that the Fc is structurally stabilized following the transition of glycans from their hybrid- to complex-type state. X-ray crystallographic analysis of this hybrid-type intermediate reveals that N-linked glycans undergo conformational changes upon maturation, including a flip within the trimannosyl core. Our crystal structure of this intermediate reveals a molecular basis for antibody biogenesis and provides a template for the structure-guided engineering of the protein-glycan interface of therapeutic antibodies.

Owen RL, Axford D, Nettleship JE, Owens RJ, Robinson JI, Morgan AW, Doré AS, Lebon G, Tate CG, Fry EE et al. 2012. Outrunning free radicals in room-temperature macromolecular crystallography. Acta Crystallogr D Biol Crystallogr, 68 (Pt 7), pp. 810-818. | Show Abstract | Read more

A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin γ Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A(2A) adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macromolecular crystallography.

Sainsbury S, Ren J, Saunders NJ, Stuart DI, Owens RJ. 2012. Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) from Neisseria meningitidis. Acta Crystallogr Sect F Struct Biol Cryst Commun, 68 (Pt 7), pp. 730-737. | Show Abstract | Read more

The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from Neisseria meningitidis, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators.

Walter TS, Ren J, Tuthill TJ, Rowlands DJ, Stuart DI, Fry EE. 2012. A plate-based high-throughput assay for virus stability and vaccine formulation. J Virol Methods, 185 (1), pp. 166-170. | Show Abstract | Read more

Standard methods for assessing the thermal stability of viruses can be time consuming and rather qualitative yet such data is a necessary requisite for vaccine formulation. In this study a novel plate-based thermal scanning assay for virus particle stability has been developed (PaSTRy: Particle Stability Thermal Release Assay). Two environment-sensitive fluorescent dyes, with non-overlapping emission spectra and different affinities, are used to accrue simultaneously independent data for the overall stability of the virus capsid, as judged by the exposure of the genome, and for capsid protein stability according to the exposure of hydrophobic side chains which are normally buried. This offers a fast and efficient high-throughput method to optimise vaccine formulation and to investigate the processes of virus uncoating.

Axford D, Owen RL, Aishima J, Foadi J, Morgan AW, Robinson JI, Nettleship JE, Owens RJ, Moraes I, Fry EE et al. 2012. In situ macromolecular crystallography using microbeams. Acta Crystallogr D Biol Crystallogr, 68 (Pt 5), pp. 592-600. | Show Abstract | Read more

Despite significant progress in high-throughput methods in macromolecular crystallography, the production of diffraction-quality crystals remains a major bottleneck. By recording diffraction in situ from crystals in their crystallization plates at room temperature, a number of problems associated with crystal handling and cryoprotection can be side-stepped. Using a dedicated goniometer installed on the microfocus macromolecular crystallography beamline I24 at Diamond Light Source, crystals have been studied in situ with an intense and flexible microfocus beam, allowing weakly diffracting samples to be assessed without a manual crystal-handling step but with good signal to noise, despite the background scatter from the plate. A number of case studies are reported: the structure solution of bovine enterovirus 2, crystallization screening of membrane proteins and complexes, and structure solution from crystallization hits produced via a high-throughput pipeline. These demonstrate the potential for in situ data collection and structure solution with microbeams.

Porta C, Xu X, Loureiro S, Paramasivam S, Ren J, Al-Khalil T, Burman A, Jackson T, Belsham GJ, Curry S et al. 2013. Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity. J Virol Methods, 187 (2), pp. 406-412. | Show Abstract | Read more

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

Sarin LP, Wright S, Chen Q, Degerth LH, Stuart DI, Grimes JM, Bamford DH, Poranen MM. 2012. The C-terminal priming domain is strongly associated with the main body of bacteriophage ϕ6 RNA-dependent RNA polymerase. Virology, 432 (1), pp. 184-193. | Show Abstract | Read more

Double-stranded RNA viruses encode a single protein species containing RNA-dependent RNA polymerase (RdRP) motifs. This protein is responsible for RNA transcription and replication. The architecture of viral RdRPs resembles that of a cupped right hand with fingers, palm and thumb domains. Those using de novo initiation have a flexible structural elaboration that constitutes the priming platform. Here we investigate the properties of the C-terminal priming domain of bacteriophage ϕ6 to get insights into the role of an extended loop connecting this domain to the main body of the polymerase. Proteolyzed ϕ6 RdRP that possesses a nick in the hinge region of this loop was better suited for de novo initiation. The clipped C-terminus remained associated with the main body of the polymerase via the anchor helix. The structurally flexible hinge region appeared to be involved in the control of priming platform movement. Moreover, we detected abortive initiation products for a bacteriophage RdRP.

Sainsbury S, Ren J, Saunders NJ, Stuart DI, Owens RJ. 2012. Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) from Neisseria meningitidis Acta Crystallographica Section F Structural Biology and Crystallization Communications, 68 (7), pp. 730-737. | Show Abstract | Read more

The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from Neisseria meningitidis, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators. © 2012 International Union of Crystallography All rights reserved.

Rissanen I, Pawlowski A, Harlos K, Grimes JM, Stuart DI, Bamford JK. 2012. Crystallization and preliminary crystallographic analysis of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. Acta Crystallogr Sect F Struct Biol Cryst Commun, 68 (Pt 5), pp. 580-583. | Show Abstract | Read more

Members of the diverse double-β-barrel lineage of viruses are identified by the conserved structure of their major coat protein. New members of this lineage have been discovered based on structural analysis and we are interested in identifying relatives that utilize unusual versions of the double-β-barrel fold. One candidate for such studies is P23-77, an icosahedral dsDNA bacteriophage that infects the extremophile Thermus thermophilus. P23-77 has two major coat proteins, namely VP16 and VP17, of a size consistent with a single-β-barrel core fold. These previously unstudied proteins have now been successfully expressed as recombinant proteins, purified and crystallized using hanging-drop and sitting-drop vapour-diffusion methods. Crystals of coat proteins VP16 and VP17 have been obtained as well as of a putative complex. In addition, virus-derived material has been crystallized. Diffraction data have been collected to beyond 3 Å resolution for five crystal types and structure determinations are in progress.

Abrescia NG, Bamford DH, Grimes JM, Stuart DI. 2012. Structure unifies the viral universe. Annu Rev Biochem, 81 (1), pp. 795-822. | Show Abstract | Read more

Is it possible to meaningfully comprehend the diversity of the viral world? We propose that it is. This is based on the observation that, although there is immense genomic variation, every infective virion is restricted by strict constraints in structure space (i.e., there are a limited number of ways to fold a protein chain, and only a small subset of these have the potential to construct a virion, the hallmark of a virus). We have previously suggested the use of structure for the higher-order classification of viruses, where genomic similarities are no longer observable. Here, we summarize the arguments behind this proposal, describe the current status of structural work, highlighting its power to infer common ancestry, and discuss the limitations and obstacles ahead of us. We also reflect on the future opportunities for a more concerted effort to provide high-throughput methods to facilitate the large-scale sampling of the virosphere.

Cited:

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Wang X, Peng W, Ren J, Hu Z, Xu J, Lou Z, Li X, Yin W, Shen X, Porta C et al. 2012. A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71 Nature Structural and Molecular Biology, 19 (4), pp. 424-429. | Show Abstract | Read more

Enterovirus 71 (EV71) is a major agent of hand, foot and mouth disease in children that can cause severe central nervous system disease and death. No vaccine or antiviral therapy is available. High-resolution structural analysis of the mature virus and natural empty particles shows that the mature virus is structurally similar to other enteroviruses. In contrast, the empty particles are markedly expanded and resemble elusive enterovirus-uncoating intermediates not previously characterized in atomic detail. Hydrophobic pockets in the EV71 capsid are collapsed in this expanded particle, providing a detailed explanation of the mechanism for receptor-binding triggered virus uncoating. These structures provide a model for enterovirus uncoating in which the VP1 GH loop acts as an adaptor-sensor for cellular receptor attachment, converting heterologous inputs to a generic uncoating mechanism, highlighting new opportunities for therapeutic intervention. © 2012 Nature America, Inc. All rights reserved.

Wang X, Peng W, Ren J, Hu Z, Xu J, Lou Z, Li X, Yin W, Shen X, Porta C et al. 2012. A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat Struct Mol Biol, 19 (4), pp. 424-429. | Show Abstract | Read more

Enterovirus 71 (EV71) is a major agent of hand, foot and mouth disease in children that can cause severe central nervous system disease and death. No vaccine or antiviral therapy is available. High-resolution structural analysis of the mature virus and natural empty particles shows that the mature virus is structurally similar to other enteroviruses. In contrast, the empty particles are markedly expanded and resemble elusive enterovirus-uncoating intermediates not previously characterized in atomic detail. Hydrophobic pockets in the EV71 capsid are collapsed in this expanded particle, providing a detailed explanation of the mechanism for receptor-binding triggered virus uncoating. These structures provide a model for enterovirus uncoating in which the VP1 GH loop acts as an adaptor-sensor for cellular receptor attachment, converting heterologous inputs to a generic uncoating mechanism, highlighting new opportunities for therapeutic intervention.

Wright S, Poranen MM, Bamford DH, Stuart DI, Grimes JM. 2012. Noncatalytic ions direct the RNA-dependent RNA polymerase of bacterial double-stranded RNA virus ϕ6 from de novo initiation to elongation. J Virol, 86 (5), pp. 2837-2849. | Show Abstract | Read more

RNA-dependent RNA polymerases (RdRps) are key to the replication of RNA viruses. A common divalent cation binding site, distinct from the positions of catalytic ions, has been identified in many viral RdRps. We have applied biochemical, biophysical, and structural approaches to show how the RdRp from bacteriophage ϕ6 uses the bound noncatalytic Mn(2+) to facilitate the displacement of the C-terminal domain during the transition from initiation to elongation. We find that this displacement releases the noncatalytic Mn(2+), which must be replaced for elongation to occur. By inserting a dysfunctional Mg(2+) at this site, we captured two nucleoside triphosphates within the active site in the absence of Watson-Crick base pairing with template and mapped movements of divalent cations during preinitiation. These structures refine the pathway from preinitiation through initiation to elongation for the RNA-dependent RNA polymerization reaction, explain the role of the noncatalytic divalent cation in 6 RdRp, and pinpoint the previously unresolved Mn(2+)-dependent step in replication.

Bahar MW, Graham SC, Stuart DI, Grimes JM. 2011. Insights into the evolution of a complex virus from the crystal structure of vaccinia virus D13. Structure, 19 (7), pp. 1011-1020. | Show Abstract | Read more

The morphogenesis of poxviruses such as vaccinia virus (VACV) sees the virion shape mature from spherical to brick-shaped. Trimeric capsomers of the VACV D13 protein form a transitory, stabilizing lattice on the surface of the initial spherical immature virus particle. The crystal structure of D13 reveals that this major scaffolding protein comprises a double β barrel "jelly-roll" subunit arranged as pseudo-hexagonal trimers. These structural features are characteristic of the major capsid proteins of a lineage of large icosahedral double-stranded DNA viruses including human adenovirus and the bacteriophages PRD1 and PM2. Structure-based phylogenetic analysis confirms that VACV belongs to this lineage, suggesting that (analogously to higher organism embryogenesis) early poxvirus morphogenesis reflects their evolution from a lineage of viruses sharing a common icosahedral ancestor.

Yu C, Crispin M, Sonnen AF, Harvey DJ, Chang VT, Evans EJ, Scanlan CN, Stuart DI, Gilbert RJ, Davis SJ. 2011. Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells. Acta Crystallogr Sect F Struct Biol Cryst Commun, 67 (Pt 7), pp. 785-789. | Show Abstract | Read more

Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3-5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3-4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a=43.9, b=51.5, c=102.9 Å and space group P2(1)2(1)2(1) that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems.

Benfield CT, Mansur DS, McCoy LE, Ferguson BJ, Bahar MW, Oldring AP, Grimes JM, Stuart DI, Graham SC, Smith GL. 2011. Mapping the IkappaB kinase beta (IKKbeta)-binding interface of the B14 protein, a vaccinia virus inhibitor of IKKbeta-mediated activation of nuclear factor kappaB. J Biol Chem, 286 (23), pp. 20727-20735. | Show Abstract | Read more

The IκB kinase (IKK) complex regulates activation of NF-κB, a critical transcription factor in mediating inflammatory and immune responses. Not surprisingly, therefore, many viruses seek to inhibit NF-κB activation. The vaccinia virus B14 protein contributes to virus virulence by binding to the IKKβ subunit of the IKK complex and preventing NF-κB activation in response to pro-inflammatory stimuli. Previous crystallographic studies showed that the B14 protein has a Bcl-2-like fold and forms homodimers in the crystal. However, multi-angle light scattering indicated that B14 is in monomer-dimer equilibrium in solution. This transient self-association suggested that the hydrophobic dimerization interface of B14 might also mediate its interaction with IKKβ, and this was investigated by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric but still co-immunoprecipitated with IKKβ and blocked both NF-κB nuclear translocation and NF-κB-dependent gene expression. Therefore, B14 homodimerization is nonessential for binding and inhibition of IKKβ. In contrast, a second monomeric mutant (F130K) neither bound IKKβ nor inhibited NF-κB-dependent gene expression, demonstrating that this residue is required for the B14-IKKβ interaction. Thus, the dimerization and IKKβ-binding interfaces overlap and lie on a surface used for protein-protein interactions in many viral and cellular Bcl-2-like proteins.

Graham SC, Sarin LP, Bahar MW, Myers RA, Stuart DI, Bamford DH, Grimes JM. 2011. The N-terminus of the RNA polymerase from infectious pancreatic necrosis virus is the determinant of genome attachment. PLoS Pathog, 7 (6), pp. e1002085. | Show Abstract | Read more

The RNA-dependent RNA polymerase VP1 of infectious pancreatic necrosis virus (IPNV) is a single polypeptide responsible for both viral RNA transcription and genome replication. Sequence analysis identifies IPNV VP1 as having an unusual active site topology. We have purified, crystallized and solved the structure of IPNV VP1 to 2.3 Å resolution in its apo form and at 2.2 Å resolution bound to the catalytically-activating metal magnesium. We find that recombinantly-expressed VP1 is highly active for RNA transcription and replication, yielding both free and polymerase-attached RNA products. IPNV VP1 also possesses terminal (deoxy)nucleotide transferase, RNA-dependent DNA polymerase (reverse transcriptase) and template-independent self-guanylylation activity. The N-terminus of VP1 interacts with the active-site cleft and we show that the N-terminal serine residue is required for formation of covalent RNA:polymerase complexes, providing a mechanism for the genesis of viral genome:polymerase complexes observed in vivo.

Daniel E, Lin B, Diprose JM, Griffiths SL, Morris C, Berry IM, Owens RJ, Blake R, Wilson KS, Stuart DI, Esnouf RM. 2011. xtalPiMS: a PiMS-based web application for the management and monitoring of crystallization trials. J Struct Biol, 175 (2), pp. 230-235. | Show Abstract | Read more

A major advance in protein structure determination has been the advent of nanolitre-scale crystallization and (in a high-throughput environment) the development of robotic systems for storing and imaging crystallization trials. Most of these trials are carried out in 96-well (or higher density) plates and managing them is a significant information management challenge. We describe xtalPiMS, a web-based application for the management and monitoring of crystallization trials. xtalPiMS has a user-interface layer based on the standards of the Protein Information Management System (PiMS) and a database layer which links the crystallization trial images to the meta-data associated with a particular crystallization trial. The user interface has been optimized for the efficient monitoring of high-throughput environments with three different automated imagers and work to support a fourth imager is in progress, but it can even be of use without robotics. The database can either be a PiMS database or a legacy database for which a suitable mapping layer has been developed.

Savitsky M, Diprose JM, Morris C, Griffiths SL, Daniel E, Lin B, Daenke S, Bishop B, Siebold C, Wilson KS et al. 2011. Recording information on protein complexes in an information management system. J Struct Biol, 175 (2), pp. 224-229. | Show Abstract | Read more

The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described.

Zhao Y, Bishop B, Clay JE, Lu W, Jones M, Daenke S, Siebold C, Stuart DI, Jones EY, Aricescu AR. 2011. Automation of large scale transient protein expression in mammalian cells. J Struct Biol, 175 (2), pp. 209-215. | Show Abstract | Read more

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI⁻ cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative.

Morris C, Pajon A, Griffiths SL, Daniel E, Savitsky M, Lin B, Diprose JM, da Silva AW, Pilicheva K, Troshin P et al. 2011. The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory. Acta Crystallogr D Biol Crystallogr, 67 (Pt 4), pp. 249-260. | Show Abstract | Read more

The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described. PiMS is a web-based Java application that uses either Postgres or Oracle as the underlying relational database-management system. PiMS is available under a free licence to all academic laboratories either for local installation or for use as a managed service.

Bahar MW, Graham SC, Chen RA, Cooray S, Smith GL, Stuart DI, Grimes JM. 2011. How vaccinia virus has evolved to subvert the host immune response. J Struct Biol, 175 (2), pp. 127-134. | Show Abstract | Read more

Viruses are obligate intracellular parasites and are some of the most rapidly evolving and diverse pathogens encountered by the host immune system. Large complicated viruses, such as poxviruses, have evolved a plethora of proteins to disrupt host immune signalling in their battle against immune surveillance. Recent X-ray crystallographic analysis of these viral immunomodulators has helped form an emerging picture of the molecular details of virus-host interactions. In this review we consider some of these immune evasion strategies as they apply to poxviruses, from a structural perspective, with specific examples from the European SPINE2-Complexes initiative. Structures of poxvirus immunomodulators reveal the capacity of viruses to mimic and compete against the host immune system, using a diverse range of structural folds that are unique or acquired from their hosts with both enhanced and unexpectedly divergent functions.

Bowden TA, Jones EY, Stuart DI. 2011. Cells under siege: viral glycoprotein interactions at the cell surface. J Struct Biol, 175 (2), pp. 120-126. | Show Abstract | Read more

As obligate parasites, viruses are required to enter and replicate within their host, a process which employs many of their proteins to hijack natural cellular processes. High resolution X-ray crystallographic analysis has proven to be an ideal method to visualize the mechanisms by which such virus-host interactions occur and has revealed the innovative capacity of viruses to adapt efficiently to their hosts. In this review, we draw upon recently elucidated paramyxovirus-, arenavirus-, and poxvirus-host protein complex crystal structures to reveal both the capacity of viruses to appropriate one component of a physiological protein-protein binding event (often modifying it to out-compete the host-protein), and the ability to utilize novel binding sites on host cell surface receptors. The structures discussed shed light on a number of biological processes ranging from viral entry to virulence and host antagonism. Drawn together they reveal the common strategies which viruses have evolved to interact with their natural host. The structures also support molecular level rationales for how viruses can be transmitted to unrelated organisms and thus pose severe health risks.

Abrescia NG, Grimes JM, Oksanen HM, Bamford JK, Bamford DH, Stuart DI. 2011. The use of low-resolution phasing followed by phase extension from 7.6 to 2.5 Å resolution with noncrystallographic symmetry to solve the structure of a bacteriophage capsid protein. Acta Crystallogr D Biol Crystallogr, 67 (Pt 3), pp. 228-232. | Show Abstract | Read more

P2, the major capsid protein of bacteriophage PM2, adopts the double β-barrel fold characteristic of the PRD1-adenoviral lineage. The 2.5 Å resolution X-ray data obtained by analysis of the two major lattices of a multiple crystal of P2 were phased by molecular replacement, using as a search model structure factors to 7.6 Å resolution obtained from electron density cut from the map of the entire PM2 virion. Phase extension to 2.5 Å resolution used solely sixfold cycling averaging and solvent flattening. This represents an atypical example of an oligomeric protein for which the structure has been determined at high resolution by bootstrapping from low-resolution initial phases.

Yu C, Sonnen AF, George R, Dessailly BH, Stagg LJ, Evans EJ, Orengo CA, Stuart DI, Ladbury JE, Ikemizu S et al. 2011. Rigid-body ligand recognition drives cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor triggering. J Biol Chem, 286 (8), pp. 6685-6696. | Show Abstract | Read more

The inhibitory T-cell surface-expressed receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which belongs to the class of cell surface proteins phosphorylated by extrinsic tyrosine kinases that also includes antigen receptors, binds the related ligands, B7-1 and B7-2, expressed on antigen-presenting cells. Conformational changes are commonly invoked to explain ligand-induced "triggering" of this class of receptors. Crystal structures of ligand-bound CTLA-4 have been reported, but not the apo form, precluding analysis of the structural changes accompanying ligand binding. The 1.8-Å resolution structure of an apo human CTLA-4 homodimer emphasizes the shared evolutionary history of the CTLA-4/CD28 subgroup of the immunoglobulin superfamily and the antigen receptors. The ligand-bound and unbound forms of both CTLA-4 and B7-1 are remarkably similar, in marked contrast to B7-2, whose binding to CTLA-4 has elements of induced fit. Isothermal titration calorimetry reveals that ligand binding by CTLA-4 is enthalpically driven and accompanied by unfavorable entropic changes. The similarity of the thermodynamic parameters determined for the interactions of CTLA-4 with B7-1 and B7-2 suggests that the binding is not highly specific, but the conformational changes observed for B7-2 binding suggest some level of selectivity. The new structure establishes that rigid-body ligand interactions are capable of triggering CTLA-4 phosphorylation by extrinsic kinase(s).

Maluquer de Motes C, Cooray S, Ren H, Almeida GM, McGourty K, Bahar MW, Stuart DI, Grimes JM, Graham SC, Smith GL. 2011. Inhibition of apoptosis and NF-κB activation by vaccinia protein N1 occur via distinct binding surfaces and make different contributions to virulence. PLoS Pathog, 7 (12), pp. e1002430. | Show Abstract | Read more

Vaccinia virus (VACV) protein N1 is an intracellular virulence factor and belongs to a family of VACV B-cell lymphoma (Bcl)-2-like proteins whose members inhibit apoptosis or activation of pro-inflammatory transcription factors, such as interferon (IFN) regulatory factor-3 (IRF-3) and nuclear factor-κB (NF-κB). Unusually, N1 inhibits both apoptosis and NF-κB activation. To understand how N1 exerts these different functions, we have mutated residues in the Bcl-2-like surface groove and at the interface used to form N1 homodimers. Mutagenesis of the surface groove abolished only the N1 anti-apoptotic activity and protein crystallography showed these mutants differed from wild-type N1 only at the site of mutation. Conversely, mutagenesis of the dimer interface converted N1 to a monomer and affected only inhibition of NF-κB activation. Collectively, these data show that N1 inhibits pro-inflammatory and pro-apoptotic signalling using independent surfaces of the protein. To determine the relative contribution of each activity to virus virulence, mutant N1 alleles were introduced into a VACV strain lacking N1 and the virulence of these viruses was analysed after intradermal and intranasal inoculation in mice. In both models, VACV containing a mutant N1 unable to inhibit apoptosis had similar virulence to wild-type virus, whereas VACV containing a mutant N1 impaired for NF-κB inhibition induced an attenuated infection similar to that of the N1-deleted virus. This indicates that anti-apoptotic activity of N1 does not drive virulence in these in vivo models, and highlights the importance of pro-inflammatory signalling in the immune response against viral infections.

Savitsky M, Diprose JM, Morris C, Griffiths SL, Daniel E, Lin B, Daenke S, Bishop B, Siebold C, Wilson KS et al. 2011. Recording information on protein complexes in an information management system Journal of Structural Biology, 175 (2), pp. 224-229. | Show Abstract | Read more

The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. © 2011 Elsevier Inc.

Bowden TA, Jones EY, Stuart DI. 2011. Cells under siege: Viral glycoprotein interactions at the cell surface Journal of Structural Biology, 175 (2), pp. 120-126. | Show Abstract | Read more

As obligate parasites, viruses are required to enter and replicate within their host, a process which employs many of their proteins to hijack natural cellular processes. High resolution X-ray crystallographic analysis has proven to be an ideal method to visualize the mechanisms by which such virus-host interactions occur and has revealed the innovative capacity of viruses to adapt efficiently to their hosts. In this review, we draw upon recently elucidated paramyxovirus-, arenavirus-, and poxvirus-host protein complex crystal structures to reveal both the capacity of viruses to appropriate one component of a physiological protein-protein binding event (often modifying it to out-compete the host-protein), and the ability to utilize novel binding sites on host cell surface receptors. The structures discussed shed light on a number of biological processes ranging from viral entry to virulence and host antagonism. Drawn together they reveal the common strategies which viruses have evolved to interact with their natural host. The structures also support molecular level rationales for how viruses can be transmitted to unrelated organisms and thus pose severe health risks. © 2011 Elsevier Inc.

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Zhao Y, Bishop B, Clay JE, Lu W, Jones M, Daenke S, Siebold C, Stuart DI, Yvonne Jones E, Radu Aricescu A. 2011. Automation of large scale transient protein expression in mammalian cells Journal of Structural Biology, 175 (2), pp. 209-215. | Show Abstract | Read more

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI- cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative. © 2011 Elsevier Inc.

Perrakis A, Daenke S, Stuart DI, Sussman JL. 2011. From SPINE to SPINE-2 complexes and beyond Journal of Structural Biology, 175 (2), pp. 105. | Read more

Daniel E, Lin B, Diprose JM, Griffiths SL, Morris C, Berry IM, Owens RJ, Blake R, Wilson KS, Stuart DI, Esnouf RM. 2011. XtalPiMS: A PiMS-based web application for the management and monitoring of crystallization trials Journal of Structural Biology, 175 (2), pp. 230-235. | Show Abstract | Read more

A major advance in protein structure determination has been the advent of nanolitre-scale crystallization and (in a high-throughput environment) the development of robotic systems for storing and imaging crystallization trials. Most of these trials are carried out in 96-well (or higher density) plates and managing them is a significant information management challenge. We describe xtalPiMS, a web-based application for the management and monitoring of crystallization trials. xtalPiMS has a user-interface layer based on the standards of the Protein Information Management System (PiMS) and a database layer which links the crystallization trial images to the meta-data associated with a particular crystallization trial. The user interface has been optimized for the efficient monitoring of high-throughput environments with three different automated imagers and work to support a fourth imager is in progress, but it can even be of use without robotics. The database can either be a PiMS database or a legacy database for which a suitable mapping layer has been developed. © 2011 Elsevier Inc.

Perrakis A, Daenke S, Stuart DI, Sussman JL. 2011. From SPINE to SPINE-2 complexes and beyond. J Struct Biol, 175 (2), pp. 105. | Read more

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Sainsbury S, Bird L, Rao V, Shepherd SM, Stuart DI, Hunter WN, Owens RJ, Ren J. 2011. Crystal structures of penicillin-binding protein 3 from pseudomonas aeruginosa: Comparison of native and antibiotic-bound forms Journal of Molecular Biology, 405 (1), pp. 173-184. | Show Abstract | Read more

We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft. The orientations of the two β-lactams in the active site and the key interactions formed between the ligands and PBP3 are similar despite differences in the two drugs, indicating a degree of flexibility in the binding site. The conserved binding mode of β-lactam-based inhibitors appears to extend to other PBPs, as suggested by a comparison of the PBP3/ceftazidime complex and the Escherichia coli PBP1b/ceftoxamine complex. Since P. aeruginosa is an important human pathogen, the structural data reveal the mode of action of the frontline antibiotic ceftazidime at the molecular level. Improved drugs to combat infections by P. aeruginosa and related Gram-negative bacteria are sought and our study provides templates to assist that process and allows us to discuss new ways of inhibiting PBPs. © 2010 Elsevier Ltd All rights reserved.

Sainsbury S, Bird L, Rao V, Shepherd SM, Stuart DI, Hunter WN, Owens RJ, Ren J. 2011. Crystal structures of penicillin-binding protein 3 from Pseudomonas aeruginosa: comparison of native and antibiotic-bound forms. J Mol Biol, 405 (1), pp. 173-184. | Show Abstract | Read more

We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft. The orientations of the two β-lactams in the active site and the key interactions formed between the ligands and PBP3 are similar despite differences in the two drugs, indicating a degree of flexibility in the binding site. The conserved binding mode of β-lactam-based inhibitors appears to extend to other PBPs, as suggested by a comparison of the PBP3/ceftazidime complex and the Escherichia coli PBP1b/ceftoxamine complex. Since P. aeruginosa is an important human pathogen, the structural data reveal the mode of action of the frontline antibiotic ceftazidime at the molecular level. Improved drugs to combat infections by P. aeruginosa and related Gram-negative bacteria are sought and our study provides templates to assist that process and allows us to discuss new ways of inhibiting PBPs.

Bowden TA, Crispin M, Jones EY, Stuart DI. 2010. Shared paramyxoviral glycoprotein architecture is adapted for diverse attachment strategies. Biochem Soc Trans, 38 (5), pp. 1349-1355. | Show Abstract | Read more

Members within the paramyxovirus subfamily Paramyxovirinae constitute a large number of highly virulent human and animal pathogens. The glycoproteins present on these viruses are responsible for mediating host cell attachment and fusion and are key targets for the design of antiviral entry inhibitors. In the present review, we discuss recent structural studies which have led to a better understanding of the various mechanisms by which different paramyxoviruses use their attachment glycoproteins to hijack specific protein and glycan cell-surface receptors to facilitate viral entry. It is observed that the paramyxovirus attachment glycoprotein consists of a conserved overall structure which includes an N-terminal six-bladed β-propeller domain which is responsible for cell receptor binding. Crystal structures of this domain from different biomedically important paramyxoviruses, including measles, Nipah, Hendra, Newcastle disease and parainfluenza viruses, alone and in complex with their functional cell-surface receptors, demonstrate three contrasting mechanisms of receptor engagement that paramyxoviruses have evolved to confer discreet protein- and glycan-receptor specificity. This structural information highlights the adaptability of the paramyxovirus attachment glycoprotein surface and the potential for the emergence of new and potentially harmful viruses in human hosts.

Aalto AP, Poranen MM, Grimes JM, Stuart DI, Bamford DH. 2010. In vitro activities of the multifunctional RNA silencing polymerase QDE-1 of Neurospora crassa. J Biol Chem, 285 (38), pp. 29367-29374. | Show Abstract | Read more

QDE-1 is an RNA- and DNA-dependent RNA polymerase that has functions in the RNA silencing and DNA repair pathways of the filamentous fungus Neurospora crassa. The crystal structure of the dimeric enzyme has been solved, and the fold of its catalytic core is related closely to that of eukaryotic DNA-dependent RNA polymerases. However, the specific activities of this multifunctional enzyme are still largely unknown. In this study, we characterized the in vitro activities of the N-terminally truncated QDE-1ΔN utilizing structure-based mutagenesis. Our results indicate that QDE-1 displays five distinct catalytic activities, which can be dissected by mutating critical amino acids or by altering reaction conditions. Our data also suggest that the RNA- and DNA-dependent activities have different modes for the initiation of RNA synthesis, which may reflect the mechanism that enables the polymerase to discriminate between template nucleic acids. Moreover, we show that QDE-1 is a highly potent terminal nucleotidyltransferase. Our results suggest that QDE-1 is able to regulate its activity mode depending on the template nucleic acid. This work extends our understanding of the biochemical properties of the QDE-1 enzyme and related RNA polymerases.

Bollati M, Alvarez K, Assenberg R, Baronti C, Canard B, Cook S, Coutard B, Decroly E, de Lamballerie X, Gould EA et al. 2010. Structure and functionality in flavivirus NS-proteins: perspectives for drug design. Antiviral Res, 87 (2), pp. 125-148. | Show Abstract | Read more

Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.

Liu C, Walter TS, Huang P, Zhang S, Zhu X, Wu Y, Wedderburn LR, Tang P, Owens RJ, Stuart DI et al. 2010. Structural and functional insights of RANKL-RANK interaction and signaling. J Immunol, 184 (12), pp. 6910-6919. | Show Abstract | Read more

Bone remodeling involves bone resorption by osteoclasts and synthesis by osteoblasts and is tightly regulated by the receptor activator of the NF-kappaB ligand (RANKL)/receptor activator of the NF-kappaB (RANK)/osteoprotegerin molecular triad. RANKL, a member of the TNF superfamily, induces osteoclast differentiation, activation and survival upon interaction with its receptor RANK. The decoy receptor osteoprotegerin inhibits osteoclast formation by binding to RANKL. Imbalance in this molecular triad can result in diseases, including osteoporosis and rheumatoid arthritis. In this study, we report the crystal structures of unliganded RANK and its complex with RANKL and elucidation of critical residues for the function of the receptor pair. RANK represents the longest TNFR with four full cysteine-rich domains (CRDs) in which the CRD4 is stabilized by a sodium ion and a rigid linkage with CRD3. On association, RANK moves via a hinge region between the CRD2 and CRD3 to make close contact with RANKL; a significant structural change previously unseen in the engagement of TNFR superfamily 1A with its ligand. The high-affinity interaction between RANK and RANKL, maintained by continuous contact between the pair rather than the patched interaction commonly observed, is necessary for the function because a slightly reduced affinity induced by mutation produces significant disruption of osteoclast formation. The structures of RANK and RANKL-RANK complex and the biological data presented in the paper are essential for not only our understanding of the specific nature of the signaling mechanism and of disease-related mutations found in patients but also structure based drug design.

Fry EE, Tuthill TJ, Harlos K, Walter TS, Rowlands DJ, Stuart DI. 2010. Crystal structure of equine rhinitis A virus in complex with its sialic acid receptor. J Gen Virol, 91 (Pt 8), pp. 1971-1977. | Show Abstract | Read more

Equine rhinitis A virus (ERAV) shares many features with foot-and-mouth disease virus (FMDV) and both are classified within the genus Aphthovirus of the family Picornaviridae. ERAV is used as a surrogate for FMDV research as it does not require high-level biosecurity. In contrast to FMDV, which uses integrins as cellular receptors, the receptor for ERAV has been reported to involve the sugar moiety sialic acid. This study confirmed the importance of sialic acid for cell entry by ERAV and reports the crystal structure of ERAV particles complexed with the receptor analogue 3'-sialyllactose. The receptor is attached to the rim of a capsid pit adjacent to the major immunogenic site and distinct from the sialic acid binding site used by a related picornavirus, the cardiovirus Theiler's murine encephalitis virus. The structure of the major antigenic determinant of the virus, previously identified from antibody escape mutations, is also described as the EF loop of VP1, which forms a hairpin stretching across the capsid surface close to the icosahedral fivefold axis, neighbouring the receptor-binding site, and spanning two protomeric units.

Sonnen AF, Yu C, Evans EJ, Stuart DI, Davis SJ, Gilbert RJ. 2010. Domain metastability: a molecular basis for immunoglobulin deposition? J Mol Biol, 399 (2), pp. 207-213. | Show Abstract | Read more

We present the crystal structure of an immunoglobulin light-chain-like domain, CTLA-4, as a strand-swapped dimer displaying cis-trans proline isomerisation and native-like hydrogen bonding. We also show that CTLA-4 can form amyloid-like fibres and amorphous deposits explainable by the same strand swapping. Our results suggest a molecular basis for the pathological aggregation of immunoglobulin domains and why amyloid-like fibres are more often composed of homologous rather than heterologous subunits.

Bowden TA, Crispin M, Harvey DJ, Jones EY, Stuart DI. 2010. Dimeric architecture of the Hendra virus attachment glycoprotein: evidence for a conserved mode of assembly. J Virol, 84 (12), pp. 6208-6217. | Show Abstract | Read more

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed beta-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to alpha-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60 degrees ) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses.

Stahl SJ, Watts NR, Rader C, DiMattia MA, Mage RG, Palmer I, Kaufman JD, Grimes JM, Stuart DI, Steven AC, Wingfield PT. 2010. Generation and characterization of a chimeric rabbit/human Fab for co-crystallization of HIV-1 Rev. J Mol Biol, 397 (3), pp. 697-708. | Show Abstract | Read more

Rev is a key regulatory protein of human immunodeficiency virus type 1. Its function is to bind to viral transcripts and effect export from the nucleus of unspliced mRNA, thereby allowing the synthesis of structural proteins. Despite its evident importance, the structure of Rev has remained unknown, primarily because Rev's proclivity for polymerization and aggregation is an impediment to crystallization. Monoclonal antibody antigen-binding domains (Fabs) have proven useful for the co-crystallization of other refractory proteins. In the present study, a chimeric rabbit/human anti-Rev Fab was selected by phage display, expressed in a bacterial secretion system, and purified from the media. The Fab readily solubilized polymeric Rev. The resulting Fab/Rev complex was purified by metal ion affinity chromatography and characterized by analytical ultracentrifugation, which demonstrated monodispersity and indicated a 1:1 molar stoichiometry. The Fab binds with very high affinity, as determined by surface plasmon resonance, to a conformational epitope in the N-terminal half of Rev. The complex forms crystals suitable for structure determination. The ability to serve as a crystallization aid is a new application of broad utility for chimeric rabbit/human Fab. The corresponding single-chain antibody (scFv) was also prepared, offering the potential of intracellular antibody therapeutics against human immunodeficiency virus type 1.

DiMattia MA, Watts NR, Stahl SJ, Rader C, Wingfield PT, Stuart DI, Steven AC, Grimes JM. 2010. Implications of the HIV-1 Rev dimer structure at 3.2 A resolution for multimeric binding to the Rev response element. Proc Natl Acad Sci U S A, 107 (13), pp. 5810-5814. | Show Abstract | Read more

HIV-1 Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process Rev oligomerizes in association with a highly structured RNA motif, the Rev response element. Crystallographic studies of Rev have been hampered by the protein's tendency to aggregate, but Rev has now been found to form a stable soluble equimolar complex with a specifically engineered monoclonal Fab fragment. We have determined the structure of this complex at 3.2 A resolution. It reveals a molecular dimer of Rev, bound on either side by a Fab, where the ordered portion of each Rev monomer (residues 9-65) contains two coplanar alpha-helices arranged in hairpin fashion. Subunits dimerize through overlapping of the hairpin prongs. Mating of hydrophobic patches on the outer surface of the dimer is likely to promote higher order interactions, suggesting a model for Rev oligomerization onto the viral RNA.

Assenberg R, Delmas O, Morin B, Graham SC, De Lamballerie X, Laubert C, Coutard B, Grimes JM, Neyts J, Owens RJ et al. 2010. Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription. Antiviral Res, 87 (2), pp. 149-161. | Show Abstract | Read more

Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication.

Bell CH, Porter SL, Strawson A, Stuart DI, Armitage JP. 2010. Using structural information to change the phosphotransfer specificity of a two-component chemotaxis signalling complex. PLoS Biol, 8 (2), pp. e1000306. | Show Abstract | Read more

Two-component signal transduction pathways comprising histidine protein kinases (HPKs) and their response regulators (RRs) are widely used to control bacterial responses to environmental challenges. Some bacteria have over 150 different two-component pathways, and the specificity of the phosphotransfer reactions within these systems is tightly controlled to prevent unwanted crosstalk. One of the best understood two-component signalling pathways is the chemotaxis pathway. Here, we present the 1.40 A crystal structure of the histidine-containing phosphotransfer domain of the chemotaxis HPK, CheA(3), in complex with its cognate RR, CheY(6). A methionine finger on CheY(6) that nestles in a hydrophobic pocket in CheA(3) was shown to be important for the interaction and was found to only occur in the cognate RRs of CheA(3), CheY(6), and CheB(2). Site-directed mutagenesis of this methionine in combination with two adjacent residues abolished binding, as shown by surface plasmon resonance studies, and phosphotransfer from CheA(3)-P to CheY(6). Introduction of this methionine and an adjacent alanine residue into a range of noncognate CheYs, dramatically changed their specificity, allowing protein interaction and rapid phosphotransfer from CheA(3)-P. The structure presented here has allowed us to identify specificity determinants for the CheA-CheY interaction and subsequently to successfully reengineer phosphotransfer signalling. In summary, our results provide valuable insight into how cells mediate specificity in one of the most abundant signalling pathways in biology, two-component signal transduction.

Sainsbury S, Ren J, Nettleship JE, Saunders NJ, Stuart DI, Owens RJ. 2010. The structure of a reduced form of OxyR from Neisseria meningitidis. BMC Struct Biol, 10 (1), pp. 10. | Show Abstract | Read more

BACKGROUND: Survival of the human pathogen, Neisseria meningitidis, requires an effective response to oxidative stress resulting from the release of hydrogen peroxide by cells of the human immune system. In N. meningitidis, expression of catalase, which is responsible for detoxifying hydrogen peroxide, is controlled by OxyR, a redox responsive LysR-type regulator. OxyR responds directly to intracellular hydrogen peroxide through the reversible formation of a disulphide bond between C199 and C208 in the regulatory domain of the protein. RESULTS: We report the first crystal structure of the regulatory domain of an OxyR protein (NMB0173 from N. meningitidis) in the reduced state i.e. with cysteines at positions 199 and 208. The protein was crystallized under reducing conditions and the structure determined to a resolution of 2.4 A. The overall fold of the Neisseria OxyR shows a high degree of similarity to the structure of a C199S mutant OxyR from E. coli, which cannot form the redox sensitive disulphide. In the neisserial structure, C199 is located at the start of helix alpha3, separated by 18 A from C208, which is positioned between helices alpha3 and alpha4. In common with other LysR-type regulators, full length OxyR proteins are known to assemble into tetramers. Modelling of the full length neisserial OxyR as a tetramer indicated that C199 and C208 are located close to the dimer-dimer interface in the assembled tetramer. The formation of the C199-C208 disulphide may thus affect the quaternary structure of the protein. CONCLUSION: Given the high level of structural similarity between OxyR from N. meningitidis and E. coli, we conclude that the redox response mechanism is likely to be similar in both species, involving the reversible formation of a disulphide between C199-C208. Modelling suggests that disulphide formation would directly affect the interface between regulatory domains in an OxyR tetramer which in turn may lead to an alteration in the spacing/orientation of the DNA-binding domains and hence the interaction of OxyR with its DNA binding sites.

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Bell CH, Porter SL, Strawson A, Stuart DI, Armitage JP. 2010. Using structural information to change the phosphotransfer specificity of a two-component chemotaxis signalling complex PLoS Biology, 8 (2), | Show Abstract | Read more

Two-component signal transduction pathways comprising histidine protein kinases (HPKs) and their response regulators (RRs) are widely used to control bacterial responses to environmental challenges. Some bacteria have over 150 different two-component pathways, and the specificity of the phosphotransfer reactions within these systems is tightly controlled to prevent unwanted crosstalk. One of the best understood two-component signalling pathways is the chemotaxis pathway. Here, we present the 1.40 A° crystal structure of the histidine-containing phosphotransfer domain of the chemotaxis HPK, CheA 3, in complex with its cognate RR, CheY6. A methionine finger on CheY6 that nestles in a hydrophobic pocket in CheA 3 was shown to be important for the interaction and was found to only occur in the cognate RRs of CheA3, CheY6, and CheB 2. Site-directed mutagenesis of this methionine in combination with two adjacent residues abolished binding, as shown by surface plasmon resonance studies, and phosphotransfer from CheA3-P to CheY6. Introduction of this methionine and an adjacent alanine residue into a range of noncognate CheYs, dramatically changed their specificity, allowing protein interaction and rapid phosphotransfer from CheA3-P. The structure presented here has allowed us to identify specificity determinants for the CheA-CheY interaction and subsequently to successfully reengineer phosphotransfer signalling. In summary, our results provide valuable insight into how cells mediate specificity in one of the most abundant signalling pathways in biology, two-component signal transduction. © 2010 Bell et al.

Ji X, Sutton G, Evans G, Axford D, Owen R, Stuart DI. 2010. How baculovirus polyhedra fit square pegs into round holes to robustly package viruses. EMBO J, 29 (2), pp. 505-514. | Show Abstract | Read more

Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles.

Assenberg R, Mastrangelo E, Walter TS, Verma A, Milani M, Owens RJ, Stuart DI, Grimes JM, Mancini EJ. 2009. Crystal structure of a novel conformational state of the flavivirus NS3 protein: implications for polyprotein processing and viral replication. J Virol, 83 (24), pp. 12895-12906. | Show Abstract | Read more

The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-A-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed.

van Boxel GI, Stewart-Jones G, Holmes S, Sainsbury S, Shepherd D, Gillespie GM, Harlos K, Stuart DI, Owens R, Jones EY. 2009. Some lessons from the systematic production and structural analysis of soluble (alpha)(beta) T-cell receptors. J Immunol Methods, 350 (1-2), pp. 14-21. | Show Abstract | Read more

T-cell receptors (TCRs) are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response. The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein. Here we report our systematic approach for generating soluble, (alpha)(beta)-TCRs, for X-ray crystallographic studies. By using small-scale expression screens, novel standardized quality control mechanisms and crystallization and imaging robots we were able to add significantly to the current TCR structural database. Our success in crystallizing both isolated TCRs and Major histocompatibility complex (MHC):TCR complexes has provided us with sufficient data to develop focused crystallization screens, which have proved generically useful for the crystallization of this family of proteins and complexes.

Bowden TA, Aricescu AR, Nettleship JE, Siebold C, Rahman-Huq N, Owens RJ, Stuart DI, Jones EY. 2009. Structural plasticity of eph receptor A4 facilitates cross-class ephrin signaling. Structure, 17 (10), pp. 1386-1397. | Show Abstract | Read more

The EphA4 tyrosine kinase cell surface receptor regulates an array of physiological processes and is the only currently known class A Eph receptor that binds both A and B class ephrins with high affinity. We have solved the crystal structure of the EphA4 ligand binding domain alone and in complex with (1) ephrinB2 and (2) ephrinA2. This set of structures shows that EphA4 has significant conformational plasticity in its ligand binding face. In vitro binding data demonstrate that it has a higher affinity for class A than class B ligands. Structural analyses, drawing on previously reported Eph receptor structures, show that EphA4 in isolation and in complex with ephrinA2 resembles other class A Eph receptors but on binding ephrinB2 assumes structural hallmarks of the class B Eph receptors. This interactive plasticity reveals EphA4 as a structural chameleon, able to adopt both A and B class Eph receptor conformations, and thus provides a molecular basis for EphA-type cross-class reactivity.

Tuthill TJ, Harlos K, Walter TS, Knowles NJ, Groppelli E, Rowlands DJ, Stuart DI, Fry EE. 2009. Equine rhinitis A virus and its low pH empty particle: clues towards an aphthovirus entry mechanism? PLoS Pathog, 5 (10), pp. e1000620. | Show Abstract | Read more

Equine rhinitis A virus (ERAV) is closely related to foot-and-mouth disease virus (FMDV), belonging to the genus Aphthovirus of the Picornaviridae. How picornaviruses introduce their RNA genome into the cytoplasm of the host cell to initiate replication is unclear since they have no lipid envelope to facilitate fusion with cellular membranes. It has been thought that the dissociation of the FMDV particle into pentameric subunits at acidic pH is the mechanism for genome release during cell entry, but this raises the problem of how transfer across the endosome membrane of the genome might be facilitated. In contrast, most other picornaviruses form 'altered' particle intermediates (not reported for aphthoviruses) thought to induce membrane pores through which the genome can be transferred. Here we show that ERAV, like FMDV, dissociates into pentamers at mildly acidic pH but demonstrate that dissociation is preceded by the transient formation of empty 80S particles which have released their genome and may represent novel biologically relevant intermediates in the aphthovirus cell entry process. The crystal structures of the native ERAV virus and a low pH form have been determined via highly efficient crystallization and data collection strategies, required due to low virus yields. ERAV is closely similar to FMDV for VP2, VP3 and part of VP4 but VP1 diverges, to give a particle with a pitted surface, as seen in cardioviruses. The low pH particle has internal structure consistent with it representing a pre-dissociation cell entry intermediate. These results suggest a unified mechanism of picornavirus cell entry.

Hatherley D, Graham SC, Harlos K, Stuart DI, Barclay AN. 2009. Structure of signal-regulatory protein alpha: a link to antigen receptor evolution. J Biol Chem, 284 (39), pp. 26613-26619. | Show Abstract | Read more

Signal-regulatory protein alpha (SIRPalpha) is a myeloid membrane receptor that interacts with the membrane protein CD47, a marker of self. We have solved the structure of the complete extracellular portion of SIRPalpha, comprising three immunoglobulin superfamily domains, by x-ray crystallography to 2.5 A resolution. These data, together with previous data on the N-terminal domain and its ligand CD47 (possessing a single immunoglobulin superfamily domain), show that the CD47-SIRPalpha interaction will span a distance of around 14 nm between interacting cells, comparable with that of an immunological synapse. The N-terminal (V-set) domain mediates binding to CD47, and the two others are found to be constant (C1-set) domains. C1-set domains are restricted to proteins involved in vertebrate antigen recognition: T cell antigen receptors, immunoglobulins, major histocompatibility complex antigens, tapasin, and beta2-microglobulin. The domains of SIRPalpha (domains 2 and 3) are structurally more similar to C1-set domains than any cell surface protein not involved in antigen recognition. This strengthens the suggestion from sequence analysis that SIRP is evolutionarily closely related to antigen recognition proteins.

Crispin M, Chang VT, Harvey DJ, Dwek RA, Evans EJ, Stuart DI, Jones EY, Lord JM, Spooner RA, Davis SJ. 2009. A human embryonic kidney 293T cell line mutated at the Golgi alpha-mannosidase II locus. J Biol Chem, 284 (32), pp. 21684-21695. | Show Abstract | Read more

Disruption of Golgi alpha-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi alpha-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi alpha-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.

Sainsbury S, Lane LA, Ren J, Gilbert RJ, Saunders NJ, Robinson CV, Stuart DI, Owens RJ. 2009. The structure of CrgA from Neisseria meningitidis reveals a new octameric assembly state for LysR transcriptional regulators. Nucleic Acids Res, 37 (14), pp. 4545-4558. | Show Abstract | Read more

LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes. The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial-host cell contact. Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings. Non-denaturing mass spectrometry analysis and analytical ultracentrifugation established that the octameric form of CrgA is the predominant species in solution in both the presence and absence of an oligonucleotide encompassing the CrgA-binding sequence. Furthermore, analysis of the isolated CrgA-DNA complex by mass spectrometry showed stabilization of a double octamer species upon DNA binding. Based on the observed structure and the mass spectrometry findings, a model is proposed in which a hexadecameric array of two CrgA oligomers binds to its DNA target site.

Bowden TA, Crispin M, Graham SC, Harvey DJ, Grimes JM, Jones EY, Stuart DI. 2009. Unusual molecular architecture of the machupo virus attachment glycoprotein. J Virol, 83 (16), pp. 8259-8265. | Show Abstract | Read more

New World arenaviruses, which cause severe hemorrhagic fever, rely upon their envelope glycoproteins for attachment and fusion into their host cell. Here we present the crystal structure of the Machupo virus GP1 attachment glycoprotein, which is responsible for high-affinity binding at the cell surface to the transferrin receptor. This first structure of an arenavirus glycoprotein shows that GP1 consists of a novel alpha/beta fold. This provides a blueprint of the New World arenavirus attachment glycoproteins and reveals a new architecture of viral attachment, using a protein fold of unknown origins.

Paesen GC, Siebold C, Dallas ML, Peers C, Harlos K, Nuttall PA, Nunn MA, Stuart DI, Esnouf RM. 2009. An ion-channel modulator from the saliva of the brown ear tick has a highly modified Kunitz/BPTI structure. J Mol Biol, 389 (4), pp. 734-747. | Show Abstract | Read more

Ra-KLP, a 75 amino acid protein secreted by the salivary gland of the brown ear tick Rhipicephalus appendiculatus has a sequence resembling those of Kunitz/BPTI proteins. We report the detection, purification and characterization of the function of Ra-KLP. In addition, determination of the three-dimensional crystal structure of Ra-KLP at 1.6 A resolution using sulphur single-wavelength anomalous dispersion reveals that much of the loop structure of classical Kunitz domains, including the protruding protease-binding loop, has been replaced by beta-strands. Even more unusually, the N-terminal portion of the polypeptide chain is pinned to the "Kunitz head" by two disulphide bridges not found in classical Kunitz/BPTI proteins. The disulphide bond pattern has been further altered by the loss of the bridge that normally stabilizes the protease-binding loop. Consistent with the conversion of this loop into a beta-strand, Ra-KLP shows no significant anti-protease activity; however, it activates maxiK channels in an in vitro system, suggesting a potential mechanism for regulating host blood supply during feeding.

Walter TS, Liu C, Huang P, Zhang S, Wedderburn LR, Gao B, Owens RJ, Stuart DI, Tang P, Ren J. 2009. Crystallization and preliminary X-ray analysis of mouse RANK and its complex with RANKL. Acta Crystallogr Sect F Struct Biol Cryst Commun, 65 (Pt 6), pp. 597-600. | Show Abstract | Read more

The interaction between the TNF-family molecule receptor activator of NF-kappaB ligand (RANKL) and its receptor RANK induces osteoclast formation, activation and survival in the process of bone remodelling. RANKL-RANK also plays critical roles in T-cell/dendritic cell communication and lymph-node formation and in a variety of pathologic conditions such as tumour-cell migration and bone metastasis. Both the ectodomain of mouse RANKL and the extracellular domain of mouse RANK have been cloned, expressed and purified. Crystals of RANK alone and of RANK in complex with RANKL have been obtained that are suitable for structure determination.

Walter BL, Armitage AE, Graham SC, de Oliveira T, Skinhøj P, Jones EY, Stuart DI, McMichael AJ, Chesebro B, Iversen AK. 2009. Functional characteristics of HIV-1 subtype C compatible with increased heterosexual transmissibility. AIDS, 23 (9), pp. 1047-1057. | Show Abstract | Read more

BACKGROUND: Despite the existence of over 50 subtypes and circulating recombinant forms of HIV-1, subtype C dominates the heterosexual pandemic causing approximately 56% of all infections. OBJECTIVE: To evaluate whether viral genetic factors may contribute to the observed subtype-C predominance. METHODS: Chimeric viruses were generated using V1-V3 envelope fragments from a subtype-A/C dually infected woman with preferential genital replication of subtype C. Viral adaptation, spread and cell fusion ability were evaluated in vitro using peripheral blood mononuclear cells and HeLa-CD4-CCR5 cell lines, sequencing and cloning. Structural modeling was performed using a crystal structure of gp120-CD4-X5. Phylogenetic analysis was done using subtype-A, subtype-B and subtype-C sequences from blood and cervix of 37 infected women and database sequences. RESULTS: We identified two envelope motifs, compact V1-V2 loops and V3-316T, which are found at high frequency throughout subtype-C evolution and affect gp120 interactions with CD4 and CCR5, respectively. When a V1-Delta5 deletion or V3-A316T was incorporated into subtype A, each increased viral fusion and spread several fold in peripheral blood mononuclear cell and cell lines with low CCR5 expression. Structural modeling suggested the formation of an additional hydrogen bond between V3 and CCR5. Moreover, we found preferential selection of HIV with 316T and/or extremely short V1-V2 loops in cervices of three women infected with subtypes A/C, B or C. CONCLUSION: As CD4-CCR5-T cells are key targets for genital HIV infection and cervical selection can favor compact V1-V2 loops and 316T, which increase viral infectivity, we propose that these conserved subtype-C motifs may contribute to transmission and spread of this subtype.

Korkhin Y, Unligil UM, Littlefield O, Nelson PJ, Stuart DI, Sigler PB, Bell SD, Abrescia NG. 2009. Evolution of complex RNA polymerases: the complete archaeal RNA polymerase structure. PLoS Biol, 7 (5), pp. e1000102. | Show Abstract | Read more

The archaeal RNA polymerase (RNAP) shares structural similarities with eukaryotic RNAP II but requires a reduced subset of general transcription factors for promoter-dependent initiation. To deepen our knowledge of cellular transcription, we have determined the structure of the 13-subunit DNA-directed RNAP from Sulfolobus shibatae at 3.35 Å resolution. The structure contains the full complement of subunits, including RpoG/Rpb8 and the equivalent of the clamp-head and jaw domains of the eukaryotic Rpb1. Furthermore, we have identified subunit Rpo13, an RNAP component in the order Sulfolobales, which contains a helix-turn-helix motif that interacts with the RpoH/Rpb5 and RpoA'/Rpb1 subunits. Its location and topology suggest a role in the formation of the transcription bubble.

Crispin M, Bowden TA, Coles CH, Harlos K, Aricescu AR, Harvey DJ, Stuart DI, Jones EY. 2009. Carbohydrate and domain architecture of an immature antibody glycoform exhibiting enhanced effector functions. J Mol Biol, 387 (5), pp. 1061-1066. | Show Abstract | Read more

Antibodies contain a conserved glycosylation site that has emerged as a target for the modulation of antibody effector functions. The crystal structure of a biosynthetic intermediate of human IgG1, bearing immature oligomannose-type glycans and reported to display increased antibody-dependent cellular cytotoxicity, demonstrates that glycan engineering can bias the Fc to an open conformation primed for receptor binding.

Sarin LP, Poranen MM, Lehti NM, Ravantti JJ, Koivunen MR, Aalto AP, van Dijk AA, Stuart DI, Grimes JM, Bamford DH. 2009. Insights into the pre-initiation events of bacteriophage phi 6 RNA-dependent RNA polymerase: towards the assembly of a productive binary complex. Nucleic Acids Res, 37 (4), pp. 1182-1192. | Show Abstract | Read more

The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3' terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage phi 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi 6 RdRP can be greatly enhanced.

Graham SC, Assenberg R, Delmas O, Verma A, Gholami A, Talbi C, Owens RJ, Stuart DI, Grimes JM, Bourhy H. 2008. Rhabdovirus matrix protein structures reveal a novel mode of self-association. PLoS Pathog, 4 (12), pp. e1000251. | Show Abstract | Read more

The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins.

Bowden TA, Crispin M, Harvey DJ, Aricescu AR, Grimes JM, Jones EY, Stuart DI. 2008. Crystal structure and carbohydrate analysis of Nipah virus attachment glycoprotein: a template for antiviral and vaccine design. J Virol, 82 (23), pp. 11628-11636. | Show Abstract | Read more

Two members of the paramyxovirus family, Nipah virus (NiV) and Hendra virus (HeV), are recent additions to a growing number of agents of emergent diseases which use bats as a natural host. Identification of ephrin-B2 and ephrin-B3 as cellular receptors for these viruses has enabled the development of immunotherapeutic reagents which prevent virus attachment and subsequent fusion. Here we present the structural analysis of the protein and carbohydrate components of the unbound viral attachment glycoprotein of NiV glycoprotein (NiV-G) at a 2.2-A resolution. Comparison with its ephrin-B2-bound form reveals that conformational changes within the envelope glycoprotein are required to achieve viral attachment. Structural differences are particularly pronounced in the 579-590 loop, a major component of the ephrin binding surface. In addition, the 236-245 loop is rather disordered in the unbound structure. We extend our structural characterization of NiV-G with mass spectrometric analysis of the carbohydrate moieties. We demonstrate that NiV-G is largely devoid of the oligomannose-type glycans that in viruses such as human immunodeficiency virus type 1 and Ebola virus influence viral tropism and the host immune response. Nevertheless, we find putative ligands for the endothelial cell lectin, LSECtin. Finally, by mapping structural conservation and glycosylation site positions from other members of the paramyxovirus family, we suggest the molecular surface involved in oligomerization. These results suggest possible pathways of virus-host interaction and strategies for the optimization of recombinant vaccines.

Poranen MM, Salgado PS, Koivunen MR, Wright S, Bamford DH, Stuart DI, Grimes JM. 2008. Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase. Nucleic Acids Res, 36 (20), pp. 6633-6644. | Show Abstract | Read more

The biological role of manganese (Mn(2+)) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn(2+) by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn(2+)-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn(2+), reduced RNA binding and a compromised elongation rate. Loss of Mn(2+) binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn(2+) coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn(2+) is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.

Kadlec J, Loureiro S, Abrescia NG, Stuart DI, Jones IM. 2008. The postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines. Nat Struct Mol Biol, 15 (10), pp. 1024-1030. | Show Abstract | Read more

Viral fusion proteins mediate the merger of host and viral membranes during cell entry for all enveloped viruses. Baculovirus glycoprotein gp64 (gp64) is unusual in promoting entry into both insect and mammalian cells and is distinct from established class I and class II fusion proteins. We report the crystal structure of its postfusion form, which explains a number of gp64's biological properties including its cellular promiscuity, identifies the fusion peptides and shows it to be the third representative of a new class (III) of fusion proteins with unexpected structural homology with vesicular stomatitis virus G and herpes simplex virus type 1 gB proteins. We show that domains of class III proteins have counterparts in both class I and II proteins, suggesting that all these viral fusion machines are structurally more related than previously thought.

Meier C, Carter LG, Sainsbury S, Mancini EJ, Owens RJ, Stuart DI, Esnouf RM. 2008. The crystal structure of UMP kinase from Bacillus anthracis (BA1797) reveals an allosteric nucleotide-binding site. J Mol Biol, 381 (5), pp. 1098-1105. | Show Abstract | Read more

Uridine monophosphate (UMP) kinase is a conserved enzyme that catalyzes the ATP-driven conversion of uridylate monophosphate into uridylate diphosphate, an essential metabolic step. In prokaryotes, the enzyme exists as a homohexamer that is regulated by various metabolites. Whereas the enzymatic mechanism of UMP kinase (UK) is well-characterized, the molecular basis of its regulation remains poorly understood. Here we report the crystal structure of UK from Bacillus anthracis (BA1797) in complex with ATP at 2.82 A resolution. It reveals that the cofactor, in addition to binding in the active sites, also interacts with separate binding pockets located near the center of the hexameric structure. The existence of such an allosteric binding site had been predicted by biochemical studies, but it was not identified in previous crystal structures of prokaryotic UKs. We show that this putative allosteric pocket is conserved across different bacterial species, suggesting that it is a feature common to bacterial UKs, and we present a structural model for the allosteric regulation of this enzyme.

Abrescia NG, Grimes JM, Kivelä HM, Assenberg R, Sutton GC, Butcher SJ, Bamford JK, Bamford DH, Stuart DI. 2008. Insights into virus evolution and membrane biogenesis from the structure of the marine lipid-containing bacteriophage PM2. Mol Cell, 31 (5), pp. 749-761. | Show Abstract | Read more

Recent, primarily structural observations indicate that related viruses, harboring no sequence similarity, infect hosts of different domains of life. One such clade of viruses, defined by common capsid architecture and coat protein fold, is the so-called PRD1-adenovirus lineage. Here we report the structure of the marine lipid-containing bacteriophage PM2 determined by crystallographic analyses of the entire approximately 45 MDa virion and of the outer coat proteins P1 and P2, revealing PM2 to be a primeval member of the PRD1-adenovirus lineage with an icosahedral shell and canonical double beta barrel major coat protein. The view of the lipid bilayer, richly decorated with membrane proteins, constitutes a rare visualization of an in vivo membrane. The viral membrane proteins P3 and P6 are organized into a lattice, suggesting a possible assembly pathway to produce the mature virus.

Sainsbury S, Ren J, Saunders NJ, Stuart DI, Owens RJ. 2008. Crystallization and preliminary X-ray analysis of CrgA, a LysR-type transcriptional regulator from pathogenic Neisseria meningitidis MC58. Acta Crystallogr Sect F Struct Biol Cryst Commun, 64 (Pt 9), pp. 797-801. | Show Abstract | Read more

Although LysR-type regulators (LTTRs) represent the largest family of transcriptional regulators in bacteria, the full-length structure of only one annotated LTTR (CbnR) has been deposited in the PDB. CrgA, a LTTR from pathogenic Neisseria meningitidis MC58, which is up-regulated upon bacterial cell contact with human epithelial cells, has been cloned, purified and crystallized. Crystals of full-length CrgA were obtained after buffer screening with a thermal shift assay and concentration with 0.2 M NDSB-256. Data were collected from two crystal forms of full-length CrgA belonging to space groups P2(1)2(1)2(1) and P2(1), diffracting to 3.0 and 3.8 A resolution and consistent with the presence of between six and ten and between ten and 20 copies of CrgA in the asymmetric unit, respectively. In addition, diffraction data were collected to 2.3 A resolution from the selenomethionine derivative of the regulatory domain of CrgA. The crystals belonged to space group P2(1) and contained two molecules in the asymmetric unit.

Graham SC, Bahar MW, Cooray S, Chen RA, Whalen DM, Abrescia NG, Alderton D, Owens RJ, Stuart DI, Smith GL, Grimes JM. 2008. Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-kappaB rather than apoptosis. PLoS Pathog, 4 (8), pp. e1000128. | Show Abstract | Read more

Vaccinia virus (VACV), the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-kappaB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 A and 2.7 A resolution, respectively. Strikingly, both these proteins adopt a Bcl-2-like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2-like proteins. Unlike cellular and viral Bcl-2-like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2-like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2-like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2-like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2-like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways.

Hatherley D, Graham SC, Turner J, Harlos K, Stuart DI, Barclay AN. 2008. Paired receptor specificity explained by structures of signal regulatory proteins alone and complexed with CD47. Mol Cell, 31 (2), pp. 266-277. | Show Abstract | Read more

CD47 is a widely distributed cell-surface protein that acts a marker of self through interactions of myeloid and neural cells. We describe the high-resolution X-ray crystallographic structures of the immunoglobulin superfamily domain of CD47 alone and in complex with the N-terminal ligand-binding domain of signal regulatory protein alpha (SIRPalpha). The unusual and convoluted interacting face of CD47, comprising the N terminus and loops at the end of the domain, intercalates with the corresponding regions in SIRPalpha. We have also determined structures of the N-terminal domains of SIRPbeta, SIRPbeta(2), and SIRPgamma; proteins that are closely related to SIRPalpha but bind CD47 with negligible or reduced affinity. These results explain the specificity of CD47 for the SIRP family of paired receptors in atomic detail. Analysis of SIRPalpha polymorphisms suggests that these, as well as the activating SIRPs, may have evolved to counteract pathogen binding to the inhibitory SIRPalpha receptor.

Bowden TA, Aricescu AR, Gilbert RJ, Grimes JM, Jones EY, Stuart DI. 2008. Structural basis of Nipah and Hendra virus attachment to their cell-surface receptor ephrin-B2. Nat Struct Mol Biol, 15 (6), pp. 567-572. | Show Abstract | Read more

Nipah and Hendra viruses are emergent paramyxoviruses, causing disease characterized by rapid onset and high mortality rates, resulting in their classification as Biosafety Level 4 pathogens. Their attachment glycoproteins are essential for the recognition of the cell-surface receptors ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3). Here we report crystal structures of both Nipah and Hendra attachment glycoproteins in complex with human EFNB2. In contrast to previously solved paramyxovirus attachment complexes, which are mediated by sialic acid interactions, the Nipah and Hendra complexes are maintained by an extensive protein-protein interface, including a crucial phenylalanine side chain on EFNB2 that fits snugly into a hydrophobic pocket on the viral protein. By analogy with the development of antivirals against sialic acid binding viruses, these results provide a structural template to target antiviral inhibition of protein-protein interactions.

Au K, Ren J, Walter TS, Harlos K, Nettleship JE, Owens RJ, Stuart DI, Esnouf RM. 2008. Structures of an alanine racemase from Bacillus anthracis (BA0252) in the presence and absence of (R)-1-aminoethylphosphonic acid (L-Ala-P). Acta Crystallogr Sect F Struct Biol Cryst Commun, 64 (Pt 5), pp. 327-333. | Show Abstract | Read more

Bacillus anthracis, the causative agent of anthrax, has been targeted by the Oxford Protein Production Facility to validate high-throughput protocols within the Structural Proteomics in Europe project. As part of this work, the structures of an alanine racemase (BA0252) in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (L-Ala-P) have determined by X-ray crystallography to resolutions of 2.1 and 1.47 A, respectively. Difficulties in crystallizing this protein were overcome by the use of reductive methylation. Alanine racemase has attracted much interest as a possible target for anti-anthrax drugs: not only is D-alanine a vital component of the bacterial cell wall, but recent studies also indicate that alanine racemase, which is accessible in the exosporium, plays a key role in inhibition of germination in B. anthracis. These structures confirm the binding mode of L-Ala-P but suggest an unexpected mechanism of inhibition of alanine racemase by this compound and could provide a basis for the design of improved alanine racemase inhibitors with potential as anti-anthrax therapies.

Moran SJ, Flanagan JF, Namy O, Stuart DI, Brierley I, Gilbert RJ. 2008. The mechanics of translocation: a molecular "spring-and-ratchet" system. Structure, 16 (5), pp. 664-672. | Show Abstract | Read more

The translation of genetic information into proteins is a fundamental process of life. Stepwise addition of amino acids to the growing polypeptide chain requires the coordinated movement of mRNA and tRNAs through the ribosome, a process known as translocation. Here, we review current understanding of the kinetics and mechanics of translocation, with particular emphasis on the structure of a functional mammalian ribosome stalled during translocation by an mRNA pseudoknot. In the context of a pseudoknot-stalled complex, the translocase EF-2 is seen to compress a hybrid-state tRNA into a strained conformation. We propose that this strain energy helps overcome the kinetic barrier to translocation and drives tRNA into the P-site, with EF-2 biasing this relaxation in one direction. The tRNA can thus be considered a molecular spring and EF-2 a Brownian ratchet in a "spring-and-ratchet" system within the translocation process.

Assenberg R, Delmas O, Graham SC, Verma A, Berrow N, Stuart DI, Owens RJ, Bourhy H, Grimes JM. 2008. Expression, purification and crystallization of a lyssavirus matrix (M) protein. Acta Crystallogr Sect F Struct Biol Cryst Commun, 64 (Pt 4), pp. 258-262. | Show Abstract | Read more

The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 A resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 56.9-57.2, c = 187.9-188.6 A, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

Coutard B, Gorbalenya AE, Snijder EJ, Leontovich AM, Poupon A, De Lamballerie X, Charrel R, Gould EA, Gunther S, Norder H et al. 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res, 78 (1), pp. 37-46. | Show Abstract | Read more

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

Kainov DE, Mancini EJ, Telenius J, Lísal J, Grimes JM, Bamford DH, Stuart DI, Tuma R. 2008. Structural basis of mechanochemical coupling in a hexameric molecular motor. J Biol Chem, 283 (6), pp. 3607-3617. | Show Abstract | Read more

The P4 protein of bacteriophage phi12 is a hexameric molecular motor closely related to superfamily 4 helicases. P4 converts chemical energy from ATP hydrolysis into mechanical work, to translocate single-stranded RNA into a viral capsid. The molecular basis of mechanochemical coupling, i.e. how small approximately 1 A changes in the ATP-binding site are amplified into nanometer scale motion along the nucleic acid, is not understood at the atomic level. Here we study in atomic detail the mechanochemical coupling using structural and biochemical analyses of P4 mutants. We show that a conserved region, consisting of superfamily 4 helicase motifs H3 and H4 and loop L2, constitutes the moving lever of the motor. The lever tip encompasses an RNA-binding site that moves along the mechanical reaction coordinate. The lever is flanked by gamma-phosphate sensors (Asn-234 and Ser-252) that report the nucleotide state of neighboring subunits and control the lever position. Insertion of an arginine finger (Arg-279) into the neighboring catalytic site is concomitant with lever movement and commences ATP hydrolysis. This ensures cooperative sequential hydrolysis that is tightly coupled to mechanical motion. Given the structural conservation, the mutated residues may play similar roles in other hexameric helicases and related molecular motors.

Zaccai NR, Carter LG, Berrow NS, Sainsbury S, Nettleship JE, Walter TS, Harlos K, Owens RJ, Wilson KS, Stuart DI, Esnouf RM. 2008. Crystal structure of a 3-oxoacyl-(acylcarrier protein) reductase (BA3989) from Bacillus anthracis at 2.4-A resolution. Proteins, 70 (2), pp. 562-567. | Read more

Structural Genomics Consortium, China Structural Genomics Consortium, Northeast Structural Genomics Consortium, Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S et al. 2008. Protein production and purification. Nat Methods, 5 (2), pp. 135-146. | Show Abstract | Read more

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

Kivelä HM, Abrescia NG, Bamford JK, Grimes JM, Stuart DI, Bamford DH. 2008. Selenomethionine labeling of large biological macromolecular complexes: probing the structure of marine bacterial virus PM2. J Struct Biol, 161 (2), pp. 204-210. | Show Abstract | Read more

There is a need for improved tools for labeling protein species within large macromolecular assemblies. Here we describe a method for the efficient selenomethionine labeling of the membrane-containing bacterial virus PM2 for structural studies. By examining potential host cells a strain was found which was auxotrophic for methionine, and by performing a multiparameter search of conditions it was possible to derive a robust protocol which simultaneously minimized the toxic effects of the selenomethionine, so that a reasonable virus yield was maintained, whilst still achieving essentially complete labeling. This has allowed us to fingerprint the protein constituents of the virus in a relatively low resolution electron density map. Such a technique can be adapted to other macromolecule complexes studied by X-ray crystallography.

Walter TS, Mancini EJ, Kadlec J, Graham SC, Assenberg R, Ren J, Sainsbury S, Owens RJ, Stuart DI, Grimes JM, Harlos K. 2008. Semi-automated microseeding of nanolitre crystallization experiments. Acta Crystallogr Sect F Struct Biol Cryst Commun, 64 (Pt 1), pp. 14-18. | Show Abstract | Read more

A simple semi-automated microseeding procedure for nanolitre crystallization experiments is described. Firstly, a microseed stock solution is made from microcrystals using a Teflon bead. A dilution series of this microseed stock is then prepared and dispensed as 100 nl droplets into 96-well crystallization plates, facilitating the incorporation of seeding into high-throughput crystallization pipelines. This basic microseeding procedure has been modified to include additive-screening and cross-seeding methods. Five examples in which these techniques have been used successfully are described.

Bahar MW, Kenyon JC, Putz MM, Abrescia NG, Pease JE, Wise EL, Stuart DI, Smith GL, Grimes JM. 2008. Structure and function of A41, a vaccinia virus chemokine binding protein. PLoS Pathog, 4 (1), pp. e5. | Show Abstract | Read more

The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 A resolution. The protein has a globular beta sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI-chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine-chemokine receptor interactions.

Mancini EJ, Assenberg R, Verma A, Walter TS, Tuma R, Grimes JM, Owens RJ, Stuart DI. 2007. Structure of the Murray Valley encephalitis virus RNA helicase at 1.9 Angstrom resolution. Protein Sci, 16 (10), pp. 2294-2300. | Show Abstract | Read more

Murray Valley encephalitis virus (MVEV), a mosquito-borne flavivirus endemic to Australia, is closely related to Japanese encephalitis virus and West Nile virus. Nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains, whose activity is central to flavivirus replication and is therefore a possible target for anti-flaviviral compounds. Cloning, purification, and crystal structure determination to 1.9 Angstrom resolution of the NS3 helicase of MVEV and characterization of its enzymatic activity is reported. Comparison with the structures of helicases from related viruses supports a possible mechanism of ATP hydrolysis-driven strand separation.

Graham SC, Bahar MW, Abrescia NG, Smith GL, Stuart DI, Grimes JM. 2007. Structure of CrmE, a virus-encoded tumour necrosis factor receptor. J Mol Biol, 372 (3), pp. 660-671. | Show Abstract | Read more

Vaccinia virus (VACV), the smallpox vaccine, encodes many proteins that subvert the host immune response. One of these, cytokine response modifier E (CrmE), is secreted by infected cells and protects these cells from apoptotic challenge by tumour necrosis factor alpha (TNFalpha). We have expressed recombinant CrmE from VACV strain Lister in Escherichia coli, shown that the purified protein is monomeric in solution and competent to bind TNFalpha, and solved the structure to 2.0 A resolution. This is the first structure of a virus-encoded tumour necrosis factor receptor (TNFR). CrmE shares significant sequence similarity with mammalian type 2 TNF receptors (TNFSFR1B, p75; TNFR type 2). The structure confirms that CrmE adopts the canonical TNFR fold but only one of the two "ligand-binding" loops of TNFRSF1A is conserved in CrmE, suggesting a mechanism for the higher affinity of poxvirus TNFRs for TNFalpha over lymphotoxin-alpha. The roles of dimerisation and pre-ligand-assembly domains (PLADs) in poxvirus and mammalian TNFR activity are discussed.

Assenberg R, Ren J, Verma A, Walter TS, Alderton D, Hurrelbrink RJ, Fuller SD, Bressanelli S, Owens RJ, Stuart DI, Grimes JM. 2007. Crystal structure of the Murray Valley encephalitis virus NS5 methyltransferase domain in complex with cap analogues. J Gen Virol, 88 (Pt 8), pp. 2228-2236. | Show Abstract | Read more

We have determined the high resolution crystal structure of the methyltransferase domain of the NS5 polypeptide from the Murray Valley encephalitis virus. This domain is unusual in having both the N7 and 2'-O methyltransferase activity required for Cap 1 synthesis. We have also determined structures for complexes of this domain with nucleotides and cap analogues providing information on cap binding, based on which we suggest a model of how the sequential methylation of the N7 and 2'-O groups of the cap may be coordinated.

Cooray S, Bahar MW, Abrescia NG, McVey CE, Bartlett NW, Chen RA, Stuart DI, Grimes JM, Smith GL. 2007. Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein. J Gen Virol, 88 (Pt 6), pp. 1656-1666. | Show Abstract | Read more

Vaccinia virus (VACV) encodes many immunomodulatory proteins, including inhibitors of apoptosis and modulators of innate immune signalling. VACV protein N1 is an intracellular homodimer that contributes to virus virulence and was reported to inhibit nuclear factor (NF)-kappaB signalling. However, analysis of NF-kappaB signalling in cells infected with recombinant viruses with or without the N1L gene showed no difference in NF-kappaB-dependent gene expression. Given that N1 promotes virus virulence, other possible functions of N1 were investigated and this revealed that N1 is an inhibitor of apoptosis in cells transfected with the N1L gene and in the context of VACV infection. In support of this finding virally expressed N1 co-precipitated with endogenous pro-apoptotic Bcl-2 proteins Bid, Bad and Bax as well as with Bad and Bax expressed by transfection. In addition, the crystal structure of N1 was solved to 2.9 A resolution (0.29 nm). Remarkably, although N1 shows no sequence similarity to cellular proteins, its three-dimensional structure closely resembles Bcl-x(L) and other members of the Bcl-2 protein family. The structure also reveals that N1 has a constitutively open surface groove similar to the grooves of other anti-apoptotic Bcl-2 proteins, which bind the BH3 motifs of pro-apoptotic Bcl-2 family members. Molecular modelling of BH3 peptides into the N1 surface groove, together with analysis of their physico-chemical properties, suggests a mechanism for the specificity of peptide recognition. This study illustrates the importance of the evolutionary conservation of structure, rather than sequence, in protein function and reveals a novel anti-apoptotic protein from orthopoxviruses.

Crispin M, Aricescu AR, Chang VT, Jones EY, Stuart DI, Dwek RA, Davis SJ, Harvey DJ. 2007. Disruption of alpha-mannosidase processing induces non-canonical hybrid-type glycosylation. FEBS Lett, 581 (10), pp. 1963-1968. | Show Abstract | Read more

Golgi alpha-mannosidase II is essential for the efficient formation of complex-type glycosylation. Here, we demonstrate that the disruption of Golgi alpha-mannosidase II activity by swainsonine in human embryonic kidney cells is capable of inducing a novel class of hybrid-type glycosylation containing a partially processed mannose moiety. The discovery of 'Man(6)-based' hybrid-type glycans reveals a broader in vivo specificity of N-acetylglucosaminyltransferase I, further defines the arm-specific tolerance of core alpha1-6 fucosyltransferase to terminal alpha1-2 mannose residues, and suggests that disruption of Golgi alpha-mannosidase II activity is capable of inducing potentially 'non-self' structures.

Paesen GC, Siebold C, Harlos K, Peacey MF, Nuttall PA, Stuart DI. 2007. A tick protein with a modified Kunitz fold inhibits human tryptase. J Mol Biol, 368 (4), pp. 1172-1186. | Show Abstract | Read more

TdPI, a tick salivary gland product related to Kunitz/BPTI proteins is a potent inhibitor of human beta-tryptase. Kinetic assays suggest that three of the four catalytic sites of tryptase are blocked by TdPI, and that the inhibition of one of these involves a peptide flanking the Kunitz head. In the course of the inhibition, tryptase cleaves TdPI at several positions. Crystal structures of the TdPI head, on its own and in complex with trypsin, reveal features that are not found in classical Kunitz/BPTI proteins and suggest the mode of interaction with tryptase. The loop of TdPI connecting the beta-sheet with the C-terminal alpha-helix is shortened, the disulphide-bridge pattern altered and N and C termini separated to produce a highly pointed molecule capable of penetrating the cramped active sites of tryptase. TdPI accumulates in the cytosolic granules of mast cells, presumably suppressing inflammation in the host animal's skin by tryptase inhibition.

Hatherley D, Harlos K, Dunlop DC, Stuart DI, Barclay AN. 2007. The structure of the macrophage signal regulatory protein alpha (SIRPalpha) inhibitory receptor reveals a binding face reminiscent of that used by T cell receptors. J Biol Chem, 282 (19), pp. 14567-14575. | Show Abstract | Read more

Signal regulatory protein (SIRP) alpha is a membrane receptor that sends inhibitory signals to myeloid cells by engagement of CD47. The high resolution x-ray structure of the N-terminal ligand binding domain shows it to have a distinctive immunoglobulin superfamily V-like fold. Site-directed mutagenesis suggests that CD47 is bound at a surface involving the BC, FG, and DE loops, which distinguishes it from other immunoglobulin superfamily surface proteins that use the faces of the fold, but resembles antigen receptors. The SIRP interaction is confined to a single domain, and its use of an extended DE loop strengthens the similarity with T cell receptor binding and the suggestion that they are closely related in evolution. The employment of loops to form the CD47-binding surface provides a mechanism for small sequence changes to modulate binding specificity, explaining the different binding properties of SIRP family members.

Crispin M, Stuart DI, Jones EY. 2007. Building meaningful models of glycoproteins. Nat Struct Mol Biol, 14 (5), pp. 354. | Read more

Chotiyarnwong P, Stewart-Jones GB, Tarry MJ, Dejnirattisai W, Siebold C, Koch M, Stuart DI, Harlos K, Malasit P, Screaton G et al. 2007. Humidity control as a strategy for lattice optimization applied to crystals of HLA-A*1101 complexed with variant peptides from dengue virus. Acta Crystallogr Sect F Struct Biol Cryst Commun, 63 (Pt 5), pp. 386-392. | Show Abstract | Read more

T-cell recognition of the antigenic peptides presented by MHC class I molecules normally triggers protective immune responses, but can result in immune enhancement of disease. Cross-reactive T-cell responses may underlie immunopathology in dengue haemorrhagic fever. To analyze these effects at the molecular level, the functional MHC class I molecule HLA-A*1101 was crystallized bound to six naturally occurring peptide variants from the dengue virus NS3 protein. The crystals contained high levels of solvent and required optimization of the cryoprotectant and dehydration protocols for each complex to yield well ordered diffraction, a process that was facilitated by the use of a free-mounting system.

Sutton G, Grimes JM, Stuart DI, Roy P. 2007. Bluetongue virus VP4 is an RNA-capping assembly line. Nat Struct Mol Biol, 14 (5), pp. 449-451. | Show Abstract | Read more

Eukaryotic organisms cap the 5' ends of their messenger RNAs by a series of four chemical reactions. Some viruses achieve this using a single molecule; the crystal structure of such an enzyme from bluetongue virus reveals an elongated modular architecture that provides a scaffold for an assemblage of active sites, two contributed by a domain of novel structure.

Gilbert RJ, Gordiyenko Y, von der Haar T, Sonnen AF, Hofmann G, Nardelli M, Stuart DI, McCarthy JE. 2007. Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex. Proc Natl Acad Sci U S A, 104 (14), pp. 5788-5793. | Show Abstract | Read more

In the process of protein synthesis, the small (40S) subunit of the eukaryotic ribosome is recruited to the capped 5' end of the mRNA, from which point it scans along the 5' untranslated region in search of a start codon. However, the 40S subunit alone is not capable of functional association with cellular mRNA species; it has to be prepared for the recruitment and scanning steps by interactions with a group of eukaryotic initiation factors (eIFs). In budding yeast, an important subset of these factors (1, 2, 3, and 5) can form a multifactor complex (MFC). Here, we describe cryo-EM reconstructions of the 40S subunit, of the MFC, and of 40S complexes with MFC factors plus eIF1A. These studies reveal the positioning of the core MFC on the 40S subunit, and show how eIF-binding induces mobility in the head and platform and reconfigures the head-platform-body relationship. This is expected to increase the accessibility of the mRNA channel, thus enabling the 40S subunit to convert to a recruitment-competent state.

Jovanovic J, Takagi J, Choulier L, Abrescia NG, Stuart DI, van der Merwe PA, Mardon HJ, Handford PA. 2007. alphaVbeta6 is a novel receptor for human fibrillin-1. Comparative studies of molecular determinants underlying integrin-rgd affinity and specificity. J Biol Chem, 282 (9), pp. 6743-6751. | Show Abstract | Read more

Human fibrillin-1, the major structural protein of connective tissue 10-12 nm microfibrils, contains multiple calcium binding epidermal growth factor-like domains interspersed with transforming growth factor beta-binding protein-like (TB) domains. TB4 contains a flexible RGD loop that mediates cell adhesion via alphaVbeta3 and alpha5beta1 integrins. This study identifies integrin alphaVbeta6 as a novel cellular receptor for fibrillin-1 with a K(d) of approximately 0.45 mum. Analyses of this interaction by surface plasmon resonance and immunocytochemistry reveal different module requirements for alphaVbeta6 activation compared with those of alphaVbeta3, suggesting that a covalent linkage of an N-terminal calcium binding epidermal growth factor-like domain to TB4 can modulate alphaV integrin binding specificity. Furthermore, our data suggest alpha5beta1 is a low affinity fibrillin-1 receptor (K(d) > 1 mum), thus providing a molecular explanation for the different alpha5beta1 distribution patterns seen when human keratinocytes and fibroblasts are plated on recombinant fibrillin fragments versus those derived from the physiological ligand fibronectin. Non-focal contact distribution of alpha5beta1 suggests that its engagement by fibrillin-1 may elicit a lesser degree and/or different type of intracellular signaling compared with that seen with a high affinity ligand.

Chang VT, Crispin M, Aricescu AR, Harvey DJ, Nettleship JE, Fennelly JA, Yu C, Boles KS, Evans EJ, Stuart DI et al. 2007. Glycoprotein structural genomics: solving the glycosylation problem. Structure, 15 (3), pp. 267-273. | Show Abstract | Read more

Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the "glycosylation problem" can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.

Meier C, Carter LG, Winter G, Owens RJ, Stuart DI, Esnouf RM. 2007. Structure of 5-formyltetrahydrofolate cyclo-ligase from Bacillus anthracis (BA4489). Acta Crystallogr Sect F Struct Biol Cryst Commun, 63 (Pt 3), pp. 168-172. | Show Abstract | Read more

Bacillus anthracis is a spore-forming bacterium and the causative agent of the disease anthrax. The Oxford Protein Production Facility has been targeting proteins from B. anthracis in order to develop high-throughput technologies within the Structural Proteomics in Europe project. As part of this work, the structure of 5-formyltetrahydrofolate cyclo-ligase (BA4489) has been determined by X-ray crystallography to 1.6 A resolution. The structure, solved in complex with magnesium-ion-bound ADP and phosphate, gives a detailed picture of the proposed catalytic mechanism of the enzyme. Chemical differences from other cyclo-ligase structures close to the active site that could be exploited to design specific inhibitors are also highlighted.

Berrow NS, Alderton D, Sainsbury S, Nettleship J, Assenberg R, Rahman N, Stuart DI, Owens RJ. 2007. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res, 35 (6), pp. e45. | Show Abstract | Read more

This article describes the construction of a set of versatile expression vectors based on the In-Fusion cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His(6)-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.

Banci L, Baumeister W, Heinemann U, Schneider G, Silman I, Stuart DI, Sussman JL. 2007. An idea whose time has come. Genome Biol, 8 (11), pp. 408. | Read more

Fogg MJ, Alzari P, Bahar M, Bertini I, Betton J-M, Burmeister WP, Cambillau C, Canard B, Carrondo MA, Coll M et al. 2006. Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens (vol 62, pg 1196, 2006) ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 62 (12), pp. 1571-1571. | Read more

Salgado PS, Koivunen MR, Makeyev EV, Bamford DH, Stuart DI, Grimes JM. 2006. The structure of an RNAi polymerase links RNA silencing and transcription. PLoS Biol, 4 (12), pp. e434. | Show Abstract | Read more

RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-A resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world.

Walter TS, Meier C, Assenberg R, Au KF, Ren J, Verma A, Nettleship JE, Owens RJ, Stuart DI, Grimes JM. 2006. Lysine methylation as a routine rescue strategy for protein crystallization. Structure, 14 (11), pp. 1617-1622. | Show Abstract | Read more

Crystallization remains a critical step in X-ray structure determination. Because it is not generally possible to rationally predict crystallization conditions, commercial screens have been developed which sample a wide range of crystallization space. While this approach has proved successful in many cases, a significant number of proteins fail to crystallize despite being soluble and monodispersed. It is established that chemical modification can facilitate the crystallization of otherwise intractable proteins. Here we describe a method for the reductive methylation of lysine residues which is simple, inexpensive, and efficient, and report on its application to ten proteins. We describe the effect of methylation on the physico-chemical properties of these proteins, and show that it led to diffraction-quality crystals from four proteins and structures for three that had hitherto proved refractory to crystallization. The method is suited to both low- and high-throughput laboratories.

Au K, Berrow NS, Blagova E, Boucher IW, Boyle MP, Brannigan JA, Carter LG, Dierks T, Folkers G, Grenha R et al. 2006. Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1267-1275. | Show Abstract | Read more

A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.

Fogg MJ, Alzari P, Bahar M, Bertini I, Betton JM, Burmeister WP, Cambillau C, Canard B, Corrondo MA, Coll M et al. 2006. Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1196-1207. | Show Abstract | Read more

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.

Beteva A, Cipriani F, Cusack S, Delageniere S, Gabadinho J, Gordon EJ, Guijarro M, Hall DR, Larsen S, Launer L et al. 2006. High-throughput sample handling and data collection at synchrotrons: embedding the ESRF into the high-throughput gene-to-structure pipeline. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1162-1169. | Show Abstract | Read more

An automatic data-collection system has been implemented and installed on seven insertion-device beamlines and a bending-magnet beamline at the ESRF (European Synchrotron Radiation Facility) as part of the SPINE (Structural Proteomics In Europe) development of an automated structure-determination pipeline. The system allows remote interaction with beamline-control systems and automatic sample mounting, alignment, characterization, data collection and processing. Reports of all actions taken are available for inspection via database modules and web services.

Burman A, Clark S, Abrescia NG, Fry EE, Stuart DI, Jackson T. 2006. Specificity of the VP1 GH loop of Foot-and-Mouth Disease virus for alphav integrins. J Virol, 80 (19), pp. 9798-9810. | Show Abstract | Read more

Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by alphavbeta6 or alphavbeta8 but not for inhibition of virus binding to alphavbeta3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for alphavbeta6. In contrast, although RGDM-containing peptides were effective for alphavbeta8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses alphavbeta8 less efficiently than alphavbeta6 as a receptor for infection. Finally, evidence is presented that shows alphavbeta3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to alphavbeta6 with a higher affinity than to alphavbeta3 and alphavbeta8.

Albeck S, Alzari P, Andreini C, Banci L, Berry IM, Bertini I, Cambillau C, Canard B, Carter L, Cohen SX et al. 2006. SPINE bioinformatics and data-management aspects of high-throughput structural biology. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1184-1195. | Show Abstract | Read more

SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.

Evans EJ, Castro MA, O'Brien R, Kearney A, Walsh H, Sparks LM, Tucknott MG, Davies EA, Carmo AM, van der Merwe PA et al. 2006. Crystal structure and binding properties of the CD2 and CD244 (2B4)-binding protein, CD48. J Biol Chem, 281 (39), pp. 29309-29320. | Show Abstract | Read more

The structural analysis of surface proteins belonging to the CD2 subset of the immunoglobulin superfamily has yielded important insights into transient cellular interactions. In mice and rats, CD2 and CD244 (2B4), which are expressed predominantly on T cells and natural killer cells, respectively, bind the same, broadly expressed ligand, CD48. Structures of CD2 and CD244 have been solved previously, and we now present the structure of the receptor-binding domain of rat CD48. The receptor-binding surface of CD48 is unusually flat, as in the case of rat CD2, and shares a high degree of electrostatic complementarity with the equivalent surface of CD2. The relatively simple arrangement of charged residues and this flat topology explain why CD48 cross-reacts with CD2 and CD244 and, in rats, with the CD244-related protein, 2B4R. Comparisons of modeled complexes of CD2 and CD48 with the complex of human CD2 and CD58 are suggestive of there being substantial plasticity in the topology of ligand binding by CD2. Thermodynamic analysis of the native CD48-CD2 interaction indicates that binding is driven by equivalent, weak enthalpic and entropic effects, in contrast to the human CD2-CD58 interaction, for which there is a large entropic barrier. Overall, the structural and biophysical comparisons of the CD2 homologues suggest that the evolutionary diversification of interacting cell surface proteins is rapid and constrained only by the requirement that binding remains weak and specific.

Meier C, Aricescu AR, Assenberg R, Aplin RT, Gilbert RJ, Grimes JM, Stuart DI. 2006. The crystal structure of ORF-9b, a lipid binding protein from the SARS coronavirus. Structure, 14 (7), pp. 1157-1165. | Show Abstract | Read more

To achieve the greatest output from their limited genomes, viruses frequently make use of alternative open reading frames, in which translation is initiated from a start codon within an existing gene and, being out of frame, gives rise to a distinct protein product. These alternative protein products are, as yet, poorly characterized structurally. Here we report the crystal structure of ORF-9b, an alternative open reading frame within the nucleocapsid (N) gene from the SARS coronavirus. The protein has a novel fold, a dimeric tent-like beta structure with an amphipathic surface, and a central hydrophobic cavity that binds lipid molecules. This cavity is likely to be involved in membrane attachment and, in mammalian cells, ORF-9b associates with intracellular vesicles, consistent with a role in the assembly of the virion. Analysis of ORF-9b and other overlapping genes suggests that they provide snapshots of the early evolution of novel protein folds.

Namy O, Moran SJ, Stuart DI, Gilbert RJ, Brierley I. 2006. A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting. Nature, 441 (7090), pp. 244-247. | Show Abstract | Read more

The triplet-based genetic code requires that translating ribosomes maintain the reading frame of a messenger RNA faithfully to ensure correct protein synthesis. However, in programmed -1 ribosomal frameshifting, a specific subversion of frame maintenance takes place, wherein the ribosome is forced to shift one nucleotide backwards into an overlapping reading frame and to translate an entirely new sequence of amino acids. This process is indispensable in the replication of numerous viral pathogens, including HIV and the coronavirus associated with severe acute respiratory syndrome, and is also exploited in the expression of several cellular genes. Frameshifting is promoted by an mRNA signal composed of two essential elements: a heptanucleotide 'slippery' sequence and an adjacent mRNA secondary structure, most often an mRNA pseudoknot. How these components operate together to manipulate the ribosome is unknown. Here we describe the observation of a ribosome-mRNA pseudoknot complex that is stalled in the process of -1 frameshifting. Cryoelectron microscopic imaging of purified mammalian 80S ribosomes from rabbit reticulocytes paused at a coronavirus pseudoknot reveals an intermediate of the frameshifting process. From this it can be seen how the pseudoknot interacts with the ribosome to block the mRNA entrance channel, compromising the translocation process and leading to a spring-like deformation of the P-site transfer RNA. In addition, we identify movements of the likely eukaryotic ribosomal helicase and confirm a direct interaction between the translocase eEF2 and the P-site tRNA. Together, the structural changes provide a mechanical explanation of how the pseudoknot manipulates the ribosome into a different reading frame.

Martinez-Picado J, Prado JG, Fry EE, Pfafferott K, Leslie A, Chetty S, Thobakgale C, Honeyborne I, Crawford H, Matthews P et al. 2006. Fitness cost of escape mutations in p24 Gag in association with control of human immunodeficiency virus type 1. J Virol, 80 (7), pp. 3617-3623. | Show Abstract | Read more

Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognition is a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues 240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation, T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope associated with immune control. Analysis of HIV type 1 gag in 206 B*57/5801-positive subjects reveals three principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination strategies to limit viral escape options from such epitopes.

Gadola SD, Koch M, Marles-Wright J, Lissin NM, Shepherd D, Matulis G, Harlos K, Villiger PM, Stuart DI, Jakobsen BK et al. 2006. Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors. J Exp Med, 203 (3), pp. 699-710. | Show Abstract | Read more

Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

Stuart DI, Grimes JM. 2006. Structural studies on orbivirus proteins and particles. Curr Top Microbiol Immunol, 309 pp. 221-244. | Show Abstract

X-ray and electron microscopy analysis of Bluetongue virus (BTV), the type species of the Orbivirus genus within the family Reoviridae, have revealed various aspects of the organisation and structure of the proteins that form the viral capsid. Orbiviruses have a segmented dsRNA genome, which imposes constraints on their structure and life cycle. The atomic structure of the BTV core particle, the key viral component which transcribes the viral mRNA within the cell cytoplasm, revealed the architecture and assembly of the major core proteins VP7 and VP3. In addition, these studies formed the basis for a plausible model for the organisation of the dsRNA viral genome and the arrangement of the viral transcriptase complex (composed of the RNA-dependent RNA polymerase, the viral capping enzyme and RNA helicase) that resides within the core particle. Electron cryo-microscopy of the viral particle has shown how the two viral proteins VP2 and VP5 are arranged to form the outer capsid, with distinct packing arrangements between them and the core protein VP7. By comparison of the outer capsid proteins of orbiviruses with those of other nonturreted members of the family Reoviridae, we are able to propose a more detailed model of these structures and possible mechanisms for cell entry. Further structural results are also discussed including the atomic structure of an N-terminal domain of nonstructural protein NS2, a protein involved in virus genome assembly and morphogenesis.

Esnouf RM, Love CA, Harlos K, Stuart DI, Jones EY. 2006. Structure determination of human semaphorin 4D as an example of the use of MAD in non-optimal cases. Acta Crystallogr D Biol Crystallogr, 62 (Pt 1), pp. 108-115. | Show Abstract | Read more

Semaphorins are an important class of signalling molecules involved in axon guidance, immune function and angiogenesis. They are characterized by having an extracellular sema domain of about 500 residues. The steps involved in the determination of the structure of human semaphorin 4D are described here as a case study of selenium MAD phasing in a difficult case with low symmetry, moderate diffraction and low selenium content. A particular feature of this study was the large number of diffraction images required to give data of sufficient quality for structure determination and these data are re-analyzed here to investigate the effects of radiation damage on eventual data quality and to suggest strategies for successful MAD phasing in similar difficult cases.

Stuart DI, Jones EY, Wilson KS, Daenke S. 2006. SPINE: Structural Proteomics IN Europe – the best of both worlds Acta Crystallographica Section D Biological Crystallography, 62 (10), pp. ii-i. | Read more

Cited:

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Scopus

Salgado PS, Koivunen MRL, Makeyev EV, Bamford DH, Stuart DI, Grimes JM. 2006. The structure of an RNAi polymerase links RNA silencing and transcription. PLoS biology, 4 (12), | Show Abstract | Read more

RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-A resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world.

Evans EJ, Jansson A, Yu C, Nilsson P, Sorensen P, Stuart DI, Davis SJ. 2005. Synaptic co-stimulatory complex formation and receptor triggering IMMUNOLOGY, 116 pp. 5-5.

Evans EJ, Esnouf RM, Manso-Sancho R, Gilbert RJC, James JR, Yu C, Hyning T, Ikemizu S, Stuart DI, Davis SJ. 2005. The structure and cross-reactivity of CD28 IMMUNOLOGY, 116 pp. 54-54.

Bamford DH, Grimes JM, Stuart DI. 2005. What does structure tell us about virus evolution? Curr Opin Struct Biol, 15 (6), pp. 655-663. | Show Abstract | Read more

Viruses are the most abundant life form and infect practically all organisms. Consequently, these obligate parasites are a major cause of human suffering and economic loss. The organization and origins of this enormous virosphere are profound open questions in biology. It has generally been considered that viruses infecting evolutionally widely separated organisms (e.g. bacteria and humans) are also distinct. However, recent research contradicts this picture. Structural analyses of virion architecture and coat protein topology have revealed unexpected similarities, not visible in sequence comparisons, suggesting a common origin for viruses that infect hosts residing in different domains of life (bacteria, archaea and eukarya).

Siebold C, Berrow N, Walter TS, Harlos K, Owens RJ, Stuart DI, Terman JR, Kolodkin AL, Pasterkamp RJ, Jones EY. 2005. High-resolution structure of the catalytic region of MICAL (molecule interacting with CasL), a multidomain flavoenzyme-signaling molecule. Proc Natl Acad Sci U S A, 102 (46), pp. 16836-16841. | Show Abstract | Read more

Semaphorins are extracellular cell guidance cues that govern cytoskeletal dynamics during neuronal and vascular development. MICAL (molecule interacting with CasL) is a multidomain cytosolic protein with a putative flavoprotein monooxygenase (MO) region required for semaphorin-plexin repulsive axon guidance. Here, we report the 1.45-A resolution crystal structure of the FAD-containing MO domain of mouse MICAL-1 (residues 1-489). The topology most closely resembles that of the NADPH-dependent flavoenzyme p-hydroxybenzoate hydroxylase (PHBH). Comparison of structures before and after reaction with NADPH reveals that, as in PHBH, the flavin ring can switch between two discrete positions. In contrast with other MOs, this conformational switch is coupled with the opening of a channel to the active site, suggestive of a protein substrate. In support of this hypothesis, distinctive structural features highlight putative protein-binding sites in suitable proximity to the active site entrance. The unusual juxtaposition of this N-terminal MO (hydroxylase) activity with the characteristics of a multiprotein-binding scaffold exhibited by the C-terminal portion of the MICALs represents a unique combination of functionality to mediate signaling.

Hartley CJ, Greenwood DR, Gilbert RJ, Masoumi A, Gordon KH, Hanzlik TN, Fry EE, Stuart DI, Scotti PD. 2005. Kelp fly virus: a novel group of insect picorna-like viruses as defined by genome sequence analysis and a distinctive virion structure. J Virol, 79 (21), pp. 13385-13398. | Show Abstract | Read more

The complete genomic sequence of kelp fly virus (KFV), originally isolated from the kelp fly, Chaetocoelopa sydneyensis, has been determined. Analyses of its genomic and structural organization and phylogeny show that it belongs to a hitherto undescribed group within the picorna-like virus superfamily. The single-stranded genomic RNA of KFV is 11,035 nucleotides in length and contains a single large open reading frame encoding a polypeptide of 3,436 amino acids with 5' and 3' untranslated regions of 384 and 343 nucleotides, respectively. The predicted amino acid sequence of the polypeptide shows that it has three regions. The N-terminal region contains sequences homologous to the baculoviral inhibitor of apoptosis repeat domain, an inhibitor of apoptosis commonly found in animals and in viruses with double-stranded DNA genomes. The second region contains at least two capsid proteins. The third region has three sequence motifs characteristic of replicase proteins of many plant and animal viruses, including a helicase, a 3C chymotrypsin-like protease, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the replicase motifs shows that KFV forms a distinct and distant taxon within the picorna-like virus superfamily. Cryoelectron microscopy and image reconstruction of KFV to a resolution of 15 A reveals an icosahedral structure, with each of its 12 fivefold vertices forming a turret from the otherwise smooth surface of the 20-A-thick capsid. The architecture of the KFV capsid is unique among the members of the picornavirus superfamily for which structures have previously been determined.

Gilbert RJ, Beales L, Blond D, Simon MN, Lin BY, Chisari FV, Stuart DI, Rowlands DJ. 2005. Hepatitis B small surface antigen particles are octahedral. Proc Natl Acad Sci U S A, 102 (41), pp. 14783-14788. | Show Abstract | Read more

The infectious component of hepatitis B (HB) virus (HBV), the Dane particle, has a diameter of approximately 44 nm and consists of a double-layered capsid particle enclosing a circular, incomplete double-stranded DNA genome. The outer capsid layer is formed from the HB surface antigen (HBsAg) and lipid, whereas the inner layer is formed from the HB core Ag assembled into an icosahedral structure. During chronic infection HBsAg is expressed in large excess as noninfectious quasispherical particles and tubules with approximately 22-nm diameter. Here, we report cryo-EM reconstructions of spherical HBsAg particles at approximately 12-A resolution. We show that the particles possess different diameters and have separated them into two predominant populations, both of which have octahedral symmetry. Despite their differing diameters, the two forms of the particle have the same mass and are built through conformational switching of the same building block, a dimer of HBsAg. We propose that this conformational switching, combined with interactions with the underlying core, leads to the formation of HBV Dane particles of different sizes, dictated by the symmetry of the icosahedral core.

Anduleit K, Sutton G, Diprose JM, Mertens PP, Grimes JM, Stuart DI. 2005. Crystal lattice as biological phenotype for insect viruses. Protein Sci, 14 (10), pp. 2741-2743. | Show Abstract | Read more

Many insect viruses survive for long periods by occlusion within robust crystalline polyhedra composed primarily of a single polyhedrin protein. We show that two different virus families form polyhedra which, despite lack of sequence similarity in the virally encoded polyhedrin protein, have identical cell constants and a body-centered cubic lattice. It is almost inconceivable that this could have arisen by chance, suggesting that the crystal lattice has been preserved because it is particularly well-suited to its function of packaging and protecting viruses.

Newman J, Egan D, Walter TS, Meged R, Berry I, Ben Jelloul M, Sussman JL, Stuart DI, Perrakis A. 2005. Towards rationalization of crystallization screening for small- to medium-sized academic laboratories: the PACT/JCSG+ strategy. Acta Crystallogr D Biol Crystallogr, 61 (Pt 10), pp. 1426-1431. | Show Abstract | Read more

A crystallization screening process is presented that was developed for a small academic laboratory. Its underlying concept is to combine sparse-matrix screening with systematic screening in a minimum number of crystallization conditions. The sparse-matrix screen is the cherry-picked combination of conditions from the Joint Center for Structural Genomics (JCSG) extended using conditions from other screens. Its aim is to maximize the coverage of crystallization parameter space with no redundancy. The systematic screen, a pH-, anion- and cation-testing (PACT) screen, aims to decouple the components of each condition and to provide information about the protein, even in the absence of crystals, rather than cover a wide crystallization space. This screening strategy is combined with nanolitre-volume dispensing hardware and a small but practical experiment-tracking system. The screens have been tested both at the NKI and in other laboratories and it is concluded that they provide a useful minimal screening strategy.

Meier C, Mancini EJ, Bamford DH, Walsh MA, Stuart DI, Grimes JM. 2005. Overcoming the false-minima problem in direct methods: structure determination of the packaging enzyme P4 from bacteriophage phi13. Acta Crystallogr D Biol Crystallogr, 61 (Pt 9), pp. 1238-1244. | Show Abstract | Read more

The problems encountered during the phasing and structure determination of the packaging enzyme P4 from bacteriophage phi13 using the anomalous signal from selenium in a single-wavelength anomalous dispersion experiment (SAD) are described. The oligomeric state of P4 in the virus is a hexamer (with sixfold rotational symmetry) and it crystallizes in space group C2, with four hexamers in the crystallographic asymmetric unit. Current state-of-the-art ab initio phasing software yielded solutions consisting of 96 atoms arranged as sixfold symmetric clusters of Se atoms. However, although these solutions showed high correlation coefficients indicative that the substructure had been solved, the resulting phases produced uninterpretable electron-density maps. Only after further analysis were correct solutions found (also of 96 atoms), leading to the eventual identification of the positions of 120 Se atoms. Here, it is demonstrated how the difficulties in finding a correct phase solution arise from an intricate false-minima problem.

Abrescia NG, Kivelä HM, Grimes JM, Bamford JK, Bamford DH, Stuart DI. 2005. Preliminary crystallographic analysis of the major capsid protein P2 of the lipid-containing bacteriophage PM2. Acta Crystallogr Sect F Struct Biol Cryst Commun, 61 (Pt 8), pp. 762-765. | Show Abstract | Read more

PM2 (Corticoviridae) is a dsDNA bacteriophage which contains a lipid membrane beneath its icosahedral capsid. In this respect it resembles bacteriophage PRD1 (Tectiviridae), although it is not known whether the similarity extends to the detailed molecular architecture of the virus, for instance the fold of the major coat protein P2. Structural analysis of PM2 has been initiated and virus-derived P2 has been crystallized by sitting-nanodrop vapour diffusion. Crystals of P2 have been obtained in space group P2(1)2(1)2, with two trimers in the asymmetric unit and unit-cell parameters a = 171.1, b = 78.7, c = 130.1 A. The crystals diffract to 4 A resolution at the ESRF BM14 beamline (Grenoble, France) and the orientation of the non-crystallographic threefold axes, the spatial relationship between the two trimers and the packing of the trimers within the unit cell have been determined. The trimers form tightly packed layers consistent with the crystal morphology, possibly recapitulating aspects of the arrangement of subunits in the virus.

Fry EE, Newman JW, Curry S, Najjam S, Jackson T, Blakemore W, Lea SM, Miller L, Burman A, King AM, Stuart DI. 2005. Structure of Foot-and-mouth disease virus serotype A10 61 alone and complexed with oligosaccharide receptor: receptor conservation in the face of antigenic variation. J Gen Virol, 86 (Pt 7), pp. 1909-1920. | Show Abstract | Read more

Foot-and-mouth disease viruses (FMDVs) target epithelial cells via integrin receptors, but can acquire the capacity to bind cell-surface heparan sulphate (or alternative receptors) on passage in cell culture. Vaccine viruses must be propagated in cell culture and, hence, some rationale for the selection of variants in this process is important. Crystal structures are available for type O, A and C viruses and also for a complex of type O strain O(1)BFS with heparin. The structure of FMDV A10(61) (a cell culture-adapted strain) complexed with heparin has now been determined. This virus has an RGSD motif in place of the otherwise conserved RGD integrin-binding motif and the potential to bind heparan sulphate (suggested by sequence analyses). FMDV A10(61) was closely similar in structure to other serotypes, deviating most in antigenic sites. The VP1 GH loop comprising the integrin-binding motif was disordered. Heparin bound at a similar site and in a similar conformation to that seen in the analogous complex with O(1)BFS, although the binding had a lower affinity and was more ionic.

Ferris NP, Abrescia NG, Stuart DI, Jackson T, Burman A, King DP, Paton DJ. 2005. Utility of recombinant integrin alpha v beta6 as a capture reagent in immunoassays for the diagnosis of foot-and-mouth disease. J Virol Methods, 127 (1), pp. 69-79. | Show Abstract | Read more

Recombinant integrin alpha v beta6 was evaluated as a capture ligand in a sandwich ELISA for the detection and serotyping of foot-and-mouth disease (FMD) virus. Our routinely applied method employs seven serotype-specific rabbit polyclonal antibodies as capture ligands and seven serotype-specific guinea pig polyclonal antibodies as detecting reagents. The recombinant integrin bound FMD virus of all seven serotypes but not that of another vesicular disease, swine vesicular disease (SVD). Considerable heterotypic cross-reactions were evident when using the integrin capture ligand in combination with guinea pig detecting antibodies but totally type-specific reactions resulted when serotype-specific monoclonal antibodies (mabs) were used instead of the guinea pig reagents. The specificity of reaction of the integrin capture/mab detector combination was superior to that of our routinely employed rabbit/guinea pig ELISA and offers an improvement for test interpretation. As a universal trapping reagent for all FMD virus serotypes the alpha v beta6 recombinant protein also has the potential for application in other test procedures for viral identification (e.g. pen-side chromatographic strip-tests, biosensors, immunocapture RT-PCR, antigenic characterization procedures and monoclonal antibody profiling of emerging field virus strains) and in antibody detection assays employed for the diagnosis of FMD.

Walter TS, Diprose JM, Mayo CJ, Siebold C, Pickford MG, Carter L, Sutton GC, Berrow NS, Brown J, Berry IM et al. 2005. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr D Biol Crystallogr, 61 (Pt 6), pp. 651-657. | Show Abstract | Read more

Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.

Evans EJ, Esnouf RM, Manso-Sancho R, Gilbert RJ, James JR, Yu C, Fennelly JA, Vowles C, Hanke T, Walse B et al. 2005. Crystal structure of a soluble CD28-Fab complex. Nat Immunol, 6 (3), pp. 271-279. | Show Abstract | Read more

Naive T cell activation requires signaling by the T cell receptor and by nonclonotypic cell surface receptors. The most important costimulatory protein is the monovalent homodimer CD28, which interacts with CD80 and CD86 expressed on antigen-presenting cells. Here we present the crystal structure of a soluble form of CD28 in complex with the Fab fragment of a mitogenic antibody. Structural comparisons redefine the evolutionary relationships of CD28-related proteins, antigen receptors and adhesion molecules and account for the distinct ligand-binding and stoichiometric properties of CD28 and the related, inhibitory homodimer CTLA-4. Cryo-electron microscopy-based comparisons of complexes of CD28 with mitogenic and nonmitogenic antibodies place new constraints on models of antibody-induced receptor triggering. This work completes the initial structural characterization of the CD28-CTLA-4-CD80-CD86 signaling system.

Laurila MR, Salgado PS, Stuart DI, Grimes JM, Bamford DH. 2005. Back-priming mode of phi6 RNA-dependent RNA polymerase. J Gen Virol, 86 (Pt 2), pp. 521-526. | Show Abstract | Read more

The RNA-dependent RNA polymerase of the double-stranded RNA bacteriophage phi6 is capable of primer-independent initiation, as are many RNA polymerases. The structure of this polymerase revealed an initiation platform, composed of a loop in the C-terminal domain (QYKW, aa 629-632), that was essential for de novo initiation. A similar element has been identified in hepatitis C virus RNA-dependent RNA polymerase. Biochemical studies have addressed the role of this platform, revealing that a mutant version can utilize a back-priming initiation mechanism, where the 3' terminus of the template adopts a hairpin-like conformation. Here, the mechanism of back-primed initiation is studied further by biochemical and structural methods.

Mayo CJ, Diprose JM, Walter TS, Berry IM, Wilson J, Owens RJ, Jones EY, Harlos K, Stuart DI, Esnouf RM. 2005. Benefits of automated crystallization plate tracking, imaging, and analysis. Structure, 13 (2), pp. 175-182. | Show Abstract | Read more

We describe the design of a database and software for managing and organizing protein crystallization data. We also outline the considerations behind the design of a fast web interface linking protein production data, crystallization images, and automated image analysis. The database and associated interfaces underpin the Oxford Protein Production Facility (OPPF) crystallization laboratory, collecting, in a routine and automatic manner, up to 100,000 images per day. Over 17 million separate images are currently held in this database. We discuss the substantial scientific benefits automated tracking, imaging, and analysis of crystallizations offers to the structural biologist: analysis of the time course of the trial and easy analysis of trials with related crystallization conditions. Features of this system address requirements common to many crystallographic laboratories that are currently setting up (semi-)automated crystallization imaging systems.

Pajon A, Ionides J, Diprose J, Fillon J, Fogh R, Ashton AW, Berman H, Boucher W, Cygler M, Deleury E et al. 2005. Design of a data model for developing laboratory information management and analysis systems for protein production. Proteins, 58 (2), pp. 278-284. | Show Abstract | Read more

Data management has emerged as one of the central issues in the high-throughput processes of taking a protein target sequence through to a protein sample. To simplify this task, and following extensive consultation with the international structural genomics community, we describe here a model of the data related to protein production. The model is suitable for both large and small facilities for use in tracking samples, experiments, and results through the many procedures involved. The model is described in Unified Modeling Language (UML). In addition, we present relational database schemas derived from the UML. These relational schemas are already in use in a number of data management projects.

Laurila MR, Salgado PS, Makeyev EV, Nettelship J, Stuart DI, Grimes JM, Bamford DH. 2005. Gene silencing pathway RNA-dependent RNA polymerase of Neurospora crassa: yeast expression and crystallization of selenomethionated QDE-1 protein. J Struct Biol, 149 (1), pp. 111-115. | Show Abstract | Read more

The RNA-dependent RNA polymerase, QDE-1, is a component of the RNA silencing pathway in Neurospora crassa. The enzymatically active carboxy-terminal fragment QDE-1 DeltaN has been expressed in Saccharomyces cerevisiae in the presence and absence of selenomethionine (SeMet). The level of SeMet incorporation was estimated by mass spectrometry to be approximately 98%. Both native and SeMet proteins were crystallized in space group P2(1) with unit cell parameters a=101.2, b=122.5, c=114.4A, beta=108.9 degrees , and 2 molecules per asymmetric unit. The native and SeMet crystals diffract to 2.3 and 3.2A, respectively, the latter are suitable for MAD structure determination.

Salgado PS, Walsh MA, Laurila MR, Stuart DI, Grimes JM. 2005. Going soft and SAD with manganese. Acta Crystallogr D Biol Crystallogr, 61 (Pt 1), pp. 108-111. | Show Abstract | Read more

SAD phasing has been revisited recently, with experiments being carried out using previously unconventional sources of anomalous signal, particularly lighter atoms and softer X-rays. A case study is reported using the 75 kDa RNA-dependent RNA polymerase of the bacteriophase phi6, which binds a Mn atom and crystallizes with three molecules in the asymmetric unit. X-ray diffraction data were collected at a wavelength of 1.89 A and although the calculated anomalous signal from the three Mn atoms was only 1.2%, SHELXD and SOLVE were able to locate these atoms. SOLVE/RESOLVE used this information to obtain SAD phases and automatically build a model for the core region of the protein, which possessed the characteristic features of the right-hand polymerase motif. These results demonstrate that with modern synchrotron beamlines and software, manganese phasing is a practical tool for solving the structure of large proteins.

Mohd Jaafar F, Attoui H, Bahar MW, Siebold C, Sutton G, Mertens PP, De Micco P, Stuart DI, Grimes JM, De Lamballerie X. 2005. The structure and function of the outer coat protein VP9 of Banna virus. Structure, 13 (1), pp. 17-28. | Show Abstract | Read more

Banna virus (BAV: genus Seadornavirus, family Reoviridae) has a double-shelled morphology similar to rotavirus and bluetongue virus. The structure of BAV outer-capsid protein VP9 was determined by X-ray crystallography at 2.6 A resolution, revealing a trimeric molecule, held together by an N-terminal helical bundle, reminiscent of coiled-coil structures found in fusion-active proteins such as HIV gp41. The major domain of VP9 contains stacked beta sheets with marked structural similarities to the receptor binding protein VP8 of rotavirus. Anti-VP9 antibodies neutralize viral infectivity, and, remarkably, pretreatment of cells with trimeric VP9 increased viral infectivity, indicating that VP9 is involved in virus attachment to cell surface and subsequent internalization. Sequence similarities were also detected between BAV VP10 and VP5 portion of rotavirus VP4, suggesting that the receptor binding and internalization apparatus, which is a single gene product activated by proteoloysis in rotavirus, is the product of two separate genome segments in BAV.

Fry EE, Stuart DI, Rowlands DJ. 2005. The structure of foot-and-mouth disease virus. Curr Top Microbiol Immunol, 288 pp. 71-101. | Show Abstract

Structural studies of foot-and-mouth disease virus (FMDV) have largely focused on the mature viral particle, providing atomic resolution images of the spherical protein capsid for a number of sero- and sub-types, structures of the highly immunogenic surface loop, Fab and GAG receptor complexes. Additionally, structures are available for a few non-structural proteins. The chapter reviews our current structural knowledge and its impact on our understanding of the virus life cycle proceeding from the mature virus through immune evasion/inactivation, cell-receptor binding and replication and alludes to future structural targets.

Hopkins AL, Ren J, Milton J, Hazen RJ, Chan JH, Stuart DI, Stammers DK. 2004. Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved drug resistance properties. 1. J Med Chem, 47 (24), pp. 5912-5922. | Show Abstract | Read more

We have used a structure-based approach to design a novel series of non-nucleoside inhibitors of HIV-1 RT (NNRTIs). Detailed analysis of a wide range of crystal structures of HIV-1 RT-NNRTI complexes together with data on drug resistance mutations has identified factors important for tight binding of inhibitors and resilience to mutations. Using this approach we have designed and synthesized a novel series of quinolone NNRTIs. Crystal structure analysis of four of these compounds in complexes with HIV-1 RT confirms the predicted binding modes. Members of this quinolone series retain high activity against the important resistance mutations in RT at Tyr181Cys and Leu100Ile.

Freeman GA, Andrews Iii CW, Hopkins AL, Lowell GS, Schaller LT, Cowan JR, Gonzales SS, Koszalka GW, Hazen RJ, Boone LR et al. 2004. Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved drug resistance properties. 2. J Med Chem, 47 (24), pp. 5923-5936. | Show Abstract | Read more

HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) are part of the combination therapy currently used to treat HIV infection. The features of a new NNRTI drug for HIV treatment must include selective potent activity against both wild-type virus as well as against mutant virus that have been selected by use of current antiretroviral treatment regimens. Based on analogy with known HIV-1 NNRTI inhibitors and modeling studies utilizing the X-ray crystal structure of inhibitors bound in the HIV-1 RT, a series of substituted 2-quinolones was synthesized and evaluated as HIV-1 inhibitors.

Abrescia NG, Cockburn JJ, Grimes JM, Sutton GC, Diprose JM, Butcher SJ, Fuller SD, San Martín C, Burnett RM, Stuart DI et al. 2004. Insights into assembly from structural analysis of bacteriophage PRD1. Nature, 432 (7013), pp. 68-74. | Show Abstract | Read more

The structure of the membrane-containing bacteriophage PRD1 has been determined by X-ray crystallography at about 4 A resolution. Here we describe the structure and location of proteins P3, P16, P30 and P31. Different structural proteins seem to have specialist roles in controlling virus assembly. The linearly extended P30 appears to nucleate the formation of the icosahedral facets (composed of trimers of the major capsid protein, P3) and acts as a molecular tape-measure, defining the size of the virus and cementing the facets together. Pentamers of P31 form the vertex base, interlocking with subunits of P3 and interacting with the membrane protein P16. The architectural similarities with adenovirus and one of the largest known virus particles PBCV-1 support the notion that the mechanism of assembly of PRD1 is scaleable and applies across the major viral lineage formed by these viruses.

Cockburn JJ, Abrescia NG, Grimes JM, Sutton GC, Diprose JM, Benevides JM, Thomas GJ, Bamford JK, Bamford DH, Stuart DI. 2004. Membrane structure and interactions with protein and DNA in bacteriophage PRD1. Nature, 432 (7013), pp. 122-125. | Show Abstract | Read more

Membranes are essential for selectively controlling the passage of molecules in and out of cells and mediating the response of cells to their environment. Biological membranes and their associated proteins present considerable difficulties for structural analysis. Although enveloped viruses have been imaged at about 9 A resolution by cryo-electron microscopy and image reconstruction, no detailed crystallographic structure of a membrane system has been described. The structure of the bacteriophage PRD1 particle, determined by X-ray crystallography at about 4 A resolution, allows the first detailed analysis of a membrane-containing virus. The architecture of the viral capsid and its implications for virus assembly are presented in the accompanying paper. Here we show that the electron density also reveals the icosahedral lipid bilayer, beneath the protein capsid, enveloping the viral DNA. The viral membrane contains about 26,000 lipid molecules asymmetrically distributed between the membrane leaflets. The inner leaflet is composed predominantly of zwitterionic phosphatidylethanolamine molecules, facilitating a very close interaction with the viral DNA, which we estimate to be packaged to a pressure of about 45 atm, factors that are likely to be important during membrane-mediated DNA translocation into the host cell. In contrast, the outer leaflet is enriched in phosphatidylglycerol and cardiolipin, which show a marked lateral segregation within the icosahedral asymmetric unit. In addition, the lipid headgroups show a surprising degree of order.

Mancini EJ, Kainov DE, Grimes JM, Tuma R, Bamford DH, Stuart DI. 2004. Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation. Cell, 118 (6), pp. 743-755. | Show Abstract | Read more

Many viruses package their genome into preformed capsids using packaging motors powered by the hydrolysis of ATP. The hexameric ATPase P4 of dsRNA bacteriophage phi12, located at the vertices of the icosahedral capsid, is such a packaging motor. We have captured crystallographic structures of P4 for all the key points along the catalytic pathway, including apo, substrate analog bound, and product bound. Substrate and product binding have been observed as both binary complexes and ternary complexes with divalent cations. These structures reveal large movements of the putative RNA binding loop, which are coupled with nucleotide binding and hydrolysis, indicating how ATP hydrolysis drives RNA translocation through cooperative conformational changes. Two distinct conformations of bound nucleotide triphosphate suggest how hydrolysis is activated by RNA binding. This provides a model for chemomechanical coupling for a prototype of the large family of hexameric helicases and oligonucleotide translocating enzymes.

Gilbert RJ, Fucini P, Connell S, Fuller SD, Nierhaus KH, Robinson CV, Dobson CM, Stuart DI. 2004. Three-dimensional structures of translating ribosomes by Cryo-EM. Mol Cell, 14 (1), pp. 57-66. | Show Abstract | Read more

Cryo-electron microscopy and image reconstruction techniques have been used to obtain three-dimensional maps for E. coli ribosomes stalled following translation of three representative proteins. Comparisons of these electron density maps, at resolutions of between 13 and 16 A, with that of a nontranslating ribosome pinpoint specific structural differences in stalled ribosomes and identify additional material, including tRNAs and mRNA. In addition, the tunnel through the large subunit, the anticipated exit route of newly synthesized proteins, is partially occluded in all the stalled ribosome structures. This observation suggests that significant segments of the nascent polypeptide chains examined here could be located within an expanded tunnel, perhaps in a rudimentary globular conformation. Such behavior could be an important aspect of the folding of at least some proteins in the cellular environment.

Lee SS, Knott V, Jovanović J, Harlos K, Grimes JM, Choulier L, Mardon HJ, Stuart DI, Handford PA. 2004. Structure of the integrin binding fragment from fibrillin-1 gives new insights into microfibril organization. Structure, 12 (4), pp. 717-729. | Show Abstract | Read more

Human fibrillin-1, the major structural protein of extracellular matrix (ECM) 10-12 nm microfibrils, is dominated by 43 calcium binding epidermal growth factor-like (cbEGF) and 7 transforming growth factor beta binding protein-like (TB) domains. Crystal structures reveal the integrin binding cbEGF22-TB4-cbEGF23 fragment of human fibrillin-1 to be a Ca(2+)-rigidified tetragonal pyramid. We suggest that other cbEGF-TB pairs within the fibrillins may adopt a similar orientation to cbEGF22-TB4. In addition, we have located a flexible RGD integrin binding loop within TB4. Modeling, cell attachment and spreading assays, immunocytochemistry, and surface plasmon resonance indicate that cbEGF22 bound to TB4 is a requirement for integrin activation and provide insight into the molecular basis of the fibrillin-1 interaction with alphaVbeta3. In light of our data, we propose a novel model for the assembly of the fibrillin microfibril and a mechanism to explain its extensibility.

Mancini EJ, Kainov DE, Wei H, Gottlieb P, Tuma R, Bamford DH, Stuart DI, Grimes JM. 2004. Production, crystallization and preliminary X-ray crystallographic studies of the bacteriophage phi 12 packaging motor. Acta Crystallogr D Biol Crystallogr, 60 (Pt 3), pp. 588-590. | Show Abstract | Read more

The hexameric ATPase P4 from bacteriophage phi 12 is responsible for packaging single-stranded genomic precursors into the viral procapsid. P4 was overexpressed in Escherichia coli and purified. Crystals of native and selenomethionine-derivatized P4 have been obtained that belong to space group I222, with half a hexamer in the asymmetric unit and unit-cell parameters a = 105.0, b = 130.5, c = 158.9 A. A second crystal form of different morphology can occur in the same crystallization drop. The second form belongs to space group P1, with four hexamers in the asymmetric unit and unit-cell parameters a = 114.9, b = 125.6, c = 153.9 A, alpha = 90.1, beta = 91.6, gamma = 90.4 degrees. Synchrotron X-ray diffraction data have been collected for the I222 and P1 crystal forms to 2.0 and 2.5 A resolution, respectively.

Sutton G, Fry E, Carter L, Sainsbury S, Walter T, Nettleship J, Berrow N, Owens R, Gilbert R, Davidson A et al. 2004. The nsp9 replicase protein of SARS-coronavirus, structure and functional insights. Structure, 12 (2), pp. 341-353. | Show Abstract | Read more

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single beta-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).

Salgado PS, Makeyev EV, Butcher SJ, Bamford DH, Stuart DI, Grimes JM. 2004. The structural basis for RNA specificity and Ca2+ inhibition of an RNA-dependent RNA polymerase. Structure, 12 (2), pp. 307-316. | Show Abstract | Read more

The RNA-dependent RNA polymerase of bacteriophage phi6 transcribes mRNA from the three segments of the dsRNA viral genome. We have cocrystallized RNA oligonucleotides with the polymerase, revealing the mode of binding of RNA templates. This binding is somewhat different from that previously seen for DNA oligomers, leading to additional RNA-protein hydrogen bonds, consistent with a preference for RNA. Activation of the RNA/polymerase complex by the addition of substrate and Mg2+ initiates a single round of reaction within the crystal to form a dead-end complex that partially collapses within the enzyme active site. By replacing Mg2+ with Ca2+, we have been able to capture the inhibited complex which shows distortion that explains the structural basis for the inhibition of such polymerases by Ca2+.

Mancini EJ, Grimes JM, Malby R, Sutton GC, Kainov DE, Juuti JT, Makeyev EV, Tuma R, Bamford DH, Stuart DI. 2003. Order and disorder in crystals of hexameric NTPases from dsRNA bacteriophages. Acta Crystallogr D Biol Crystallogr, 59 (Pt 12), pp. 2337-2341. | Show Abstract | Read more

The packaging of genomic RNA in members of the Cystoviridae is performed by P4, a hexameric protein with NTPase activity. Across family members such as Phi6, Phi8 and Phi13, the P4 proteins show low levels of sequence identity, but presumably have similar atomic structures. Initial structure-determination efforts for P4 from Phi6 and Phi8 were hampered by difficulties in obtaining crystals that gave ordered diffraction. Diffraction from crystals of full-length P4 showed a variety of disorder and anisotropy. Subsequently, crystals of Phi13 P4 were obtained which yielded well ordered diffraction to 1.7 A. Comparison of the packing arrangements of P4 hexamers in different crystal forms and analysis of the disorder provides insights into the flexibility of this family of proteins, which might be an integral part of their biological function.

Love CA, Harlos K, Mavaddat N, Davis SJ, Stuart DI, Jones EY, Esnouf RM. 2003. The ligand-binding face of the semaphorins revealed by the high-resolution crystal structure of SEMA4D. Nat Struct Biol, 10 (10), pp. 843-848. | Show Abstract | Read more

Semaphorins, proteins characterized by an extracellular sema domain, regulate axon guidance, immune function and angiogenesis. The crystal structure of SEMA4D (residues 1-657) shows the sema topology to be a seven-bladed beta-propeller, revealing an unexpected homology with integrins. The sema beta-propeller contains a distinctive 77-residue insertion between beta-strands C and D of blade 5. Blade 7 is followed by a domain common to plexins, semaphorins and integrins (PSI domain), which forms a compact cysteine knot abutting the side of the propeller, and an Ig-like domain. The top face of the beta-propeller presents prominent loops characteristic of semaphorins. In addition to limited contact between the Ig-like domains, the homodimer is stabilized through extensive interactions between the top faces in a sector of the beta-propeller used for heterodimerization in integrins. This face of the propeller also mediates ligand binding in integrins, and functional data for semaphorin-receptor interactions map to the equivalent surface.

Stewart-Jones GB, McMichael AJ, Bell JI, Stuart DI, Jones EY. 2003. A structural basis for immunodominant human T cell receptor recognition. Nat Immunol, 4 (7), pp. 657-663. | Show Abstract | Read more

The anti-influenza CD8+ T cell response in HLA-A2-positive adults is almost exclusively directed at residues 58-66 of the virus matrix protein (MP(58-66)). V(beta)17V(alpha)10.2 T cell receptors (TCRs) containing a conserved arginine-serine-serine sequence in complementarity determining region 3 (CDR3) of the V(beta) segment dominate this response. To investigate the molecular basis of immunodominant selection in an outbred population, we have determined the crystal structure of V(beta)17V(alpha)10.2 in complex with MP(58-66)-HLA-A2 at a resolution of 1.4 A. We show that, whereas the TCR typically fits over an exposed side chain of the peptide, in this structure MP(58-66) exposes only main chain atoms. This distinctive orientation of V(beta)17V(alpha)10.2, which is almost orthogonal to the peptide-binding groove of HLA-A2, facilitates insertion of the conserved arginine in V(beta) CDR3 into a notch in the surface of MP(58-66)-HLA-A2. This previously unknown binding mode underlies the immunodominant T cell response.

Fry EE, Knowles NJ, Newman JW, Wilsden G, Rao Z, King AM, Stuart DI. 2003. Crystal structure of Swine vesicular disease virus and implications for host adaptation. J Virol, 77 (9), pp. 5475-5486. | Show Abstract | Read more

Swine vesicular disease virus (SVDV) is an Enterovirus of the family Picornaviridae that causes symptoms indistinguishable from those of foot-and-mouth disease virus. Phylogenetic studies suggest that it is a recently evolved genetic sublineage of the important human pathogen coxsackievirus B5 (CBV5), and in agreement with this, it has been shown to utilize the coxsackie and adenovirus receptor (CAR) for cell entry. The 3.0-A crystal structure of strain UK/27/72 SVDV (highly virulent) reveals the expected similarity in core structure to those of other picornaviruses, showing most similarity to the closest available structure to CBV5, that of coxsackievirus B3 (CBV3). Features that help to cement together and rigidify the protein subunits are extended in this virus, perhaps explaining its extreme tolerance of environmental factors. Using the large number of capsid sequences available for both SVDV and CBV5, we have mapped the amino acid substitutions that may have occurred during the supposed adaptation of SVDV to a new host onto the structure of SVDV and a model of the SVDV/CAR complex generated by reference to the cryo-electron microscopy-visualized complex of CBV3 and CAR. The changes fall into three clusters as follows: one lines the fivefold pore, a second maps to the CAR-binding site and partially overlaps the site for decay accelerating factor (DAF) to bind to echovirus 7 (ECHO7), and the third lies close to the fivefold axis, where the low-density lipoprotein receptor binds to the minor group of rhinoviruses. Later changes in SVDV (post-1971) map to the first two clusters and may, by optimizing recognition of a pig CAR and/or DAF homologue, have improved the adaptation of the virus to pigs.

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Walter TS, Diprose J, Brown J, Pickford M, Owens RJ, Stuart DI, Harlos K. 2003. A procedure for setting up high-throughput nanolitre crystallization experiments. I. Protocol design and validation JOURNAL OF APPLIED CRYSTALLOGRAPHY, 36 (2), pp. 308-314. | Show Abstract | Read more

A protocol for setting up nanolitre sitting-drop vapour-diffusion experiments is reported. The procedure uses standard crystallization screening kits and 96-well crystallization plates. Reservoir solutions are transferred from 96-deep-well blocks to crystallization plates in a single step with a Robbins-Hydra pipettor. Nanolitre droplets of protein as well as reservoir solution are dispensed by a Cartesian pipetting instrument. Experiments have been carried out to characterize the performance of this instrument. Adaptations to the Cartesian, which include an anti-evaporation cover plate, are described and tested. The protocol was designed for a high-throughput facility, but can be used in any standard crystallography laboratory.

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Brown J, Walter TS, Carter L, Abrescia NGA, Aricescu AR, Batuwangala TD, Bird LE, Brown N, Chamberlain PP, Davis SJ et al. 2003. A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results JOURNAL OF APPLIED CRYSTALLOGRAPHY, 36 (2), pp. 315-318. | Show Abstract | Read more

An initial tranche of results from day-to-day use of a robotic system for setting up 100 nl-scale vapour-diffusion sitting-drop protein crystallizations has been surveyed. The database of over 50 unrelated samples represents a snapshot of projects currently at the stage of crystallization trials in Oxford research groups and as such encompasses a broad range of proteins. The results indicate that the nanolitre-scale methodology consistently identifies more crystallization conditions than traditional hand-pipetting-style methods; however, in a number of cases successful scale-up is then problematic. Crystals grown in the initial 100 nl-scale drops have in the majority of cases allowed useful characterization of x-ray diffraction, either in-house or at synchrotron beamlines. For a significant number of projects, full x-ray diffraction data sets have been collected to 3 Å resolution or better (either in-house or at the synchrotron) from crystals grown at the 100 nl scale. To date, five structures have been determined by molecular replacement directly from such data and a further three from scale-up of conditions established at the nanolitre scale.

Cockburn JJ, Bamford JK, Grimes JM, Bamford DH, Stuart DI. 2003. Crystallization of the membrane-containing bacteriophage PRD1 in quartz capillaries by vapour diffusion. Acta Crystallogr D Biol Crystallogr, 59 (Pt 3), pp. 538-540. | Show Abstract | Read more

Crystals of bacteriophage PRD1, a virus containing an internal lipid bilayer, have been grown in thin-walled quartz capillary tubes by vapour diffusion as a means of eliminating mechanical handling of the crystals during data collection. It has been found that the addition of polyethylene glycol 20 000 (PEG 20K) to the mother liquor that bathes the crystals allows far higher resolution diffraction intensities to be observed. Growing and treating the crystals in this way has produced a small number of crystals which are particularly amenable to X-ray diffraction analysis.

Bird LE, Chamberlain PP, Stewart-Jones GB, Ren J, Stuart DI, Stammers DK. 2003. Cloning, expression, purification, and crystallisation of HIV-2 reverse transcriptase. Protein Expr Purif, 27 (1), pp. 12-18. | Show Abstract | Read more

A purification procedure is described for the isolation of recombinant HIV-2 reverse transcriptase expressed in Escherichia coli. The p68 subunit is expressed, in the absence of induction, and use of a heparin-Sepharose column produces substantially pure protein. Concentration of the homodimeric p68 reverse transcriptase pool, followed by incubation at room temperature for several days, results in full conversion by E. coli proteases to the heterodimer (p68/p55). This extended incubation simplifies the purification process and improves the yield of heterodimeric reverse transcriptase, which shows a truncation of the smaller subunit to 427 residues. The protein is then purified further by hydroxyapatite and gel-filtration chromatography to homogeneity. The HIV-2 RT is active and has been used to produce crystals that diffract to beyond 3.0 A.

Gilbert RJ, Grimes JM, Stuart DI. 2003. Hybrid vigor: hybrid methods in viral structure determination. Adv Protein Chem, 64 pp. 37-91. | Read more

Jackson T, King AM, Stuart DI, Fry E. 2003. Structure and receptor binding. Virus Res, 91 (1), pp. 33-46. | Read more

Ren J, Bird LE, Chamberlain PP, Stewart-Jones GB, Stuart DI, Stammers DK. 2002. Structure of HIV-2 reverse transcriptase at 2.35-A resolution and the mechanism of resistance to non-nucleoside inhibitors. Proc Natl Acad Sci U S A, 99 (22), pp. 14410-14415. | Show Abstract | Read more

The HIV-2 serotype of HIV is a cause of disease in parts of the West African population, and there is evidence for its spread to Europe and Asia. HIV-2 reverse transcriptase (RT) demonstrates an intrinsic resistance to non-nucleoside RT inhibitors (NNRTIs), one of two classes of anti-AIDS drugs that target the viral RT. We report the crystal structure of HIV-2 RT to 2.35 A resolution, which reveals molecular details of the resistance to NNRTIs. HIV-2 RT has a similar overall fold to HIV-1 RT but has structural differences within the "NNRTI pocket" at both conserved and nonconserved residues. The structure points to the role of sequence differences that can give rise to unfavorable inhibitor contacts or destabilization of part of the binding pocket at positions 101, 106, 138, 181, 188, and 190. We also present evidence that the conformation of Ile-181 compared with the HIV-1 Tyr-181 could be a significant contributory factor to this inherent drug resistance of HIV-2 to NNRTIs. The availability of a refined structure of HIV-2 RT will provide a stimulus for the structure-based design of novel non-nucleoside inhibitors that could be used against HIV-2 infection.

Lang HL, Jacobsen H, Ikemizu S, Andersson C, Harlos K, Madsen L, Hjorth P, Sondergaard L, Svejgaard A, Wucherpfennig K et al. 2002. A functional and structural basis for TCR cross-reactivity in multiple sclerosis. Nat Immunol, 3 (10), pp. 940-943. | Show Abstract | Read more

The multiple sclerosis (MS)-associated HLA major histocompatibility complex (MHC) class II alleles DRB1*1501, DRB5*0101 and DQB1*0602 are in strong linkage disequilibrium, making it difficult to determine which is the principal MS risk gene. Here we show that together the DRB1 and DRB5 loci may influence susceptibility to MS. We demonstrate that a T cell receptor (TCR) from an MS patient recognized both a DRB1*1501-restricted myelin basic protein (MBP) and DRB5*0101-restricted Epstein-Barr virus (EBV) peptide. Crystal structure determination of the DRB5*0101-EBV peptide complex revealed a marked degree of structural equivalence to the DRB1*1501-MBP peptide complex at the surface presented for TCR recognition. This provides structural evidence for molecular mimicry involving HLA molecules. The structural details suggest an explanation for the preponderance of MHC class II associations in HLA-associated diseases.

Chamberlain PP, Ren J, Nichols CE, Douglas L, Lennerstrand J, Larder BA, Stuart DI, Stammers DK. 2002. Crystal structures of Zidovudine- or Lamivudine-resistant human immunodeficiency virus type 1 reverse transcriptases containing mutations at codons 41, 184, and 215. J Virol, 76 (19), pp. 10015-10019. | Show Abstract | Read more

Six structures of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing combinations of resistance mutations for zidovudine (AZT) (M41L and T215Y) or lamivudine (M184V) have been determined as inhibitor complexes. Minimal conformational changes in the polymerase or nonnucleoside RT inhibitor sites compared to the mutant RTMC (D67N, K70R, T215F, and K219N) are observed, indicating that such changes may occur only with certain combinations of mutations. Model building M41L and T215Y into HIV-1 RT-DNA and docking in ATP that is utilized in the pyrophosphorolysis reaction for AZT resistance indicates that some conformational rearrangement appears necessary in RT for ATP to interact simultaneously with the M41L and T215Y mutations.

Diprose JM, Grimes JM, Sutton GC, Burroughs JN, Meyer A, Maan S, Mertens PP, Stuart DI. 2002. The core of bluetongue virus binds double-stranded RNA. J Virol, 76 (18), pp. 9533-9536. | Show Abstract | Read more

Double-stranded RNA (dsRNA) viruses conceal their genome from the host to avoid triggering unfavorable cellular responses. The crystal structure of the core of one such virus, bluetongue virus, reveals an outer surface festooned with dsRNA. This may represent a deliberate strategy to sequester dsRNA released from damaged particles to prevent host cell shutoff.

Stuart AD, McKee TA, Williams PA, Harley C, Shen S, Stuart DI, Brown TD, Lea SM. 2002. Determination of the structure of a decay accelerating factor-binding clinical isolate of echovirus 11 allows mapping of mutants with altered receptor requirements for infection. J Virol, 76 (15), pp. 7694-7704. | Show Abstract | Read more

We have used X-ray crystallography to determine the structure of a decay accelerating factor (DAF)-binding, clinic-derived isolate of echovirus 11 (EV11-207). The structures of the capsid proteins closely resemble those of capsid proteins of other picornaviruses. The structure allows us to interpret a series of amino acid changes produced by passaging EV11-207 in different cell lines as highlighting the locations of multiple receptor-binding sites on the virion surface. We suggest that a DAF-binding site is located at the fivefold axes of the virion, while the binding site for a distinct but as yet unidentified receptor is located within the canyon surrounding the virion fivefold axes.

Bamford JK, Cockburn JJ, Diprose J, Grimes JM, Sutton G, Stuart DI, Bamford DH. 2002. Diffraction quality crystals of PRD1, a 66-MDa dsDNA virus with an internal membrane. J Struct Biol, 139 (2), pp. 103-112. | Show Abstract | Read more

It has proved difficult to obtain well diffracting single crystals of macromolecular complexes rich in lipid. We report here the path that has led to crystals of the bacteriophage PRD1, a particle containing approximately 2,000 protein subunits from 18 different protein species, around 10 of which are integral membrane proteins associated with a host-derived lipid bilayer of some 12,500 lipid molecules. These crystals are capable of diffracting X-rays to Bragg spacings below 4A. It is hoped that some lessons learned from PRD1 will be applicable to other lipidic systems and that these crystals will allow, as a proof of principle, the determination of the structure of the virus in terms of a detailed atomic model.

Collins AV, Brodie DW, Gilbert RJ, Iaboni A, Manso-Sancho R, Walse B, Stuart DI, van der Merwe PA, Davis SJ. 2002. The interaction properties of costimulatory molecules revisited. Immunity, 17 (2), pp. 201-210. | Show Abstract | Read more

B7-1 and B7-2 are generally thought to have comparable structures and affinities for their receptors, CD28 and CTLA-4, each of which is assumed to be bivalent. We show instead (1) that B7-2 binds the two receptors more weakly than B7-1, (2) that, relative to its CTLA-4 binding affinity, B7-2 binds CD28 2- to 3-fold more effectively than B7-1, (3) that, unlike B7-1, B7-2 does not self-associate, and (4) that, in contrast to CTLA-4 homodimers, which are bivalent, CD28 homodimers are monovalent. Our results indicate that B7-1 markedly favors CTLA-4 over CD28 engagement, whereas B7-2 exhibits much less bias. We propose that the distinct structures and binding properties of B7-1 and B7-2 account for their overlapping but distinct effects on T cell responses.

Hon WC, Wilson MI, Harlos K, Claridge TD, Schofield CJ, Pugh CW, Maxwell PH, Ratcliffe PJ, Stuart DI, Jones EY. 2002. Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL. Nature, 417 (6892), pp. 975-978. | Show Abstract | Read more

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that controls cellular and systemic homeostatic responses to oxygen availability. HIF-1 alpha is the oxygen-regulated subunit of HIF-1, an alpha beta heterodimeric complex. HIF-1 alpha is stable in hypoxia, but in the presence of oxygen it is targeted for proteasomal degradation by the ubiquitination complex pVHL, the protein of the von Hippel Lindau (VHL) tumour suppressor gene and a component of an E3 ubiquitin ligase complex. Capture of HIF-1 alpha by pVHL is regulated by hydroxylation of specific prolyl residues in two functionally independent regions of HIF-1 alpha. The crystal structure of a hydroxylated HIF-1 alpha peptide bound to VCB (pVHL, elongins C and B) and solution binding assays reveal a single, conserved hydroxyproline-binding pocket in pVHL. Optimized hydrogen bonding to the buried hydroxyprolyl group confers precise discrimination between hydroxylated and unmodified prolyl residues. This mechanism provides a new focus for development of therapeutic agents to modulate cellular responses to hypoxia.

Bamford DH, Burnett RM, Stuart DI. 2002. Evolution of viral structure. Theor Popul Biol, 61 (4), pp. 461-470. | Show Abstract | Read more

Viruses vastly outnumber their host cells and must present a huge selective pressure. It is also becoming evident that only a small percent of the eukaryotic genome codes for molecules involved in cellular structures and functions, and that much of the remainder may have a viral origin. Viruses clearly play a central role in the biosphere, but how is this viral world organized? Classification was originally based on virus morphology and the particular host infected, but now there is an increasing trend to rely on sequence information. The type of genome (e.g., RNA or DNA, single- or double-stranded) provides fundamental classification criteria, while sequence comparisons can provide fine mapping for closely related viruses. However, it is currently very difficult to identify long-range evolutionary relationships. We present here a different approach, based on the idea that each virus has an innate "self." When the structures and functions characteristic of this "self" are identified, then they uncover relationships beyond those accessible from sequence information alone. The new approach is illustrated by sketching some possible viral lineages. We propose that urviruses were present before the division of cellular life into its current domains, and that the viral world has lineages that can be traced back to the root of the universal tree of life.

Hatanaka H, Iourin O, Rao Z, Fry E, Kingsman A, Stuart DI. 2002. Structure of equine infectious anemia virus matrix protein. J Virol, 76 (4), pp. 1876-1883. | Show Abstract | Read more

The Gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the N-terminal membrane-binding domain forming the matrix protein (MA). The 2.8-A resolution crystal structure of MA of equine infectious anemia virus (EIAV), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the MAs of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). However, unlike the structures formed by HIV-1 and SIV MAs, the oligomerization state observed is not trimeric. We discuss the potential of this molecule for membrane binding in the light of conformational differences between EIAV MA and HIV or SIV MA.

Stammers DK, Ren J, Nichols CE, Chamberlain PP, Weaver KL, Short SA, Stuart DI. 2002. Crystal structures of HIV-1 reverse transcriptase mutated at codons 100, 106 and 108 show nevirapine resistance is mediated via perturbation of interactions with Tyr181 or Tyr188 ANTIVIRAL THERAPY, 7 pp. S30-S30.

Esnouf RM, Stuart DI, Wilson KS. 2002. High-throughput structure determination Acta Crystallographica Section D: Biological Crystallography, 58 (11),

Diprose JM, Burroughs JN, Sutton GC, Goldsmith A, Gouet P, Malby R, Overton I, Ziéntara S, Mertens PP, Stuart DI, Grimes JM. 2001. Translocation portals for the substrates and products of a viral transcription complex: the bluetongue virus core. EMBO J, 20 (24), pp. 7229-7239. | Show Abstract | Read more

The bluetongue virus core is a molecular machine that simultaneously and repeatedly transcribes mRNA from 10 segments of viral double-stranded RNA, packaged in a liquid crystalline array. To determine how the logistical problems of transcription within a sealed shell are solved, core crystals were soaked with various ligands and analysed by X-ray crystallography. Mg(2+) ions produce a slight expansion of the capsid around the 5-fold axes. Oligonucleotide soaks demonstrate that the 5-fold pore, opened up by this expansion, is the exit site for mRNA, whilst nucleotide soaks pinpoint a separate binding site that appears to be a selective channel for the entry and exit of substrates and by-products. Finally, nucleotides also bind to the outer core layer, providing a substrate sink.

Maenaka K, van der Merwe PA, Stuart DI, Jones EY, Sondermann P. 2001. The human low affinity Fcgamma receptors IIa, IIb, and III bind IgG with fast kinetics and distinct thermodynamic properties. J Biol Chem, 276 (48), pp. 44898-44904. | Show Abstract | Read more

Fcgamma receptors (FcgammaRs) are expressed on all immunologically active cells. They bind the Fc portion of IgG, thereby triggering a range of immunological functions. We have used surface plasmon resonance to analyze the kinetic and thermodynamic properties of the interactions between the ectodomains of human low affinity FcgammaRs (FcgammaRIIa, FcgammaRIIb, and FcgammaRIIIb-NA2) and IgG1 or the Fc fragment of IgG1. All three receptors bind Fc or IgG with similarly low affinities (K(D) approximately 0.6-2.5 microm) and fast kinetics, suggesting that FcgammaR-mediated recognition of aggregated IgG and IgG-coated particles or cells is mechanistically similar to cell-cell recognition. Interestingly, the Fc receptors exhibit distinct thermodynamic properties. Whereas the binding of the FcgammaRIIa and FcgammaRIIb to Fc is driven by favorable entropic and enthalpic changes, the binding of FcgammaRIII is characterized by highly unfavorable entropic changes. Although the structural bases for these differences remain to be determined, they suggest that the molecular events coupled to the binding differ among the low affinity FcgammaRs.

Ren J, Nichols C, Bird L, Chamberlain P, Weaver K, Short S, Stuart DI, Stammers DK. 2001. Structural mechanisms of drug resistance for mutations at codons 181 and 188 in HIV-1 reverse transcriptase and the improved resilience of second generation non-nucleoside inhibitors. J Mol Biol, 312 (4), pp. 795-805. | Show Abstract | Read more

Mutations at either Tyr181 or Tyr188 within HIV-1 reverse transcriptase (RT) give high level resistance to many first generation non-nucleoside inhibitors (NNRTIs) such as the anti-AIDS drug nevirapine. By comparison second generation inhibitors, for instance the drug efavirenz, show much greater resilience to these mutations. In order to understand the structural basis for these differences we have determined a series of seven crystal structures of mutant RTs in complexes with first and second generation NNRTIs as well as one example of an unliganded mutant RT. These are Tyr181Cys RT (TNK-651) to 2.4 A, Tyr181Cys RT (efavirenz) to 2.6 A, Tyr181Cys RT (nevirapine) to 3.0 A, Tyr181Cys RT (PETT-2) to 3.0 A, Tyr188Cys RT (nevirapine) to 2.6 A, Tyr188Cys RT (UC-781) to 2.6 A and Tyr188Cys RT (unliganded) to 2.8 A resolution. In the two previously published structures of HIV-1 reverse transcriptase with mutations at 181 or 188 no side-chain electron density was observed within the p66 subunit (which contains the inhibitor binding pocket) for the mutated residues. In contrast the mutated side-chains can be seen in the NNRTI pocket for all seven structures reported here, eliminating the possibility that disordering contributes to the mechanism of resistance. In the case of the second generation compounds efavirenz with Tyr181Cys RT and UC-781 with Tyr188Cys RT there are only small rearrangements of either inhibitor within the binding site compared to wild-type RT and also for the first generation compounds TNK-651, PETT-2 and nevirapine with Tyr181Cys RT. For nevirapine with the Tyr188Cys RT there is however a more substantial movement of the drug molecule. We conclude that protein conformational changes and rearrangements of drug molecules within the mutated sites are not general features of these particular inhibitor/mutant combinations. The main contribution to drug resistance for Tyr181Cys and Tyr188Cys RT mutations is the loss of aromatic ring stacking interactions for first generation compounds, providing a simple explanation for the resilience of second generation NNRTIs, as such interactions make much less significant contribution to their binding.

Chan JH, Hong JS, Hunter RN, Orr GF, Cowan JR, Sherman DB, Sparks SM, Reitter BE, Andrews CW, Hazen RJ et al. 2001. 2-Amino-6-arylsulfonylbenzonitriles as non-nucleoside reverse transcriptase inhibitors of HIV-1. J Med Chem, 44 (12), pp. 1866-1882. | Show Abstract | Read more

A series of 2-amino-5-arylthiobenzonitriles (1) was found to be active against HIV-1. Structural modifications led to the sulfoxides (2) and sulfones (3). The sulfoxides generally showed antiviral activity against HIV-1 similar to that of 1. The sulfones, however, were the most potent series of analogues, a number having activity against HIV-1 in the nanomolar range. Structural-activity relationship (SAR) studies suggested that a meta substituent, particularly a meta methyl substituent, invariably increased antiviral activities. However, optimal antiviral activities were manifested by compounds where both meta groups in the arylsulfonyl moiety were substituted and one of the substituents was a methyl group. Such a disubstitution led to compounds 3v, 3w, 3x, and 3y having IC50 values against HIV-1 in the low nanomolar range. When gauged for their broad-spectrum antiviral activity against key non-nucleoside reverse transcriptase inhibitor (NNRTI) related mutants, all the di-meta-substituted sulfones 3u-z and the 2-naphthyl analogue 3ee generally showed single-digit nanomolar activity against the V106A and P236L strains and submicromolar to low nanomolar activity against strains E138K, V108I, and Y188C. However, they showed a lack of activity against the K103N and Y181C mutant viruses. The elucidation of the X-ray crystal structure of the complex of 3v (739W94) in HIV-1 reverse transcriptase showed an overlap in the binding domain when compared with the complex of nevirapine in HIV-1 reverse transcriptase. The X-ray structure allowed for the rationalization of SAR data and potencies of the compounds against the mutants.

Davis SJ, Ikemizu S, Collins AV, Fennelly JA, Harlos K, Jones EY, Stuart DI. 2001. Crystallization and functional analysis of a soluble deglycosylated form of the human costimulatory molecule B7-1. Acta Crystallogr D Biol Crystallogr, 57 (Pt 4), pp. 605-608. | Show Abstract | Read more

The interactions of B7-1 with CD28 and CTLA-4 modulate the course of human immune responses, making B7-1 an important target for developing structure-based therapeutics. B7-1 is, however, one of the most heavily glycosylated proteins found at the leukocyte cell surface, complicating the structural analysis of this molecule. Methods for the production, crystallization and selenomethionine labelling of a soluble deglycosylated form of this molecule are described. The protein readily forms both tetragonal plate and bipyramidal crystals belonging to space groups I4(1)22, with unit-cell parameters a = b = 56.9, c = 298.7 A, and P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 89.0, c = 261.9 A, respectively. The I4(1)22 and primitive crystal forms diffract to 2.7 and 3.5 A, respectively. Surface plasmon resonance-based assays indicate that the ligand-binding properties of sB7-1 are unaffected by deglycosylation. Since none of the methods relied on any special structural properties of sB7-1, it is proposed that this novel combination of procedures could in principle be adapted to the systematic analysis of many other glycoproteins of structural or functional interest.

Butcher SJ, Grimes JM, Makeyev EV, Bamford DH, Stuart DI. 2001. A mechanism for initiating RNA-dependent RNA polymerization. Nature, 410 (6825), pp. 235-240. | Show Abstract | Read more

In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase. Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA. Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail. Here we show that the 2 A resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage straight phi6 is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route. The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction. Our results provide a working model for the initiation of replication and transcription.

Bamford DH, Gilbert RJ, Grimes JM, Stuart DI. 2001. Macromolecular assemblies: greater than their parts. Curr Opin Struct Biol, 11 (1), pp. 107-113. | Show Abstract | Read more

Increasingly powerful methods of analysis have opened up complex macromolecular assemblies to scrutiny at atomic detail. They reveal not only examples of assembly from preformed and prefolded components, but also examples in which the act of assembly drives changes to the components. In the most extreme of these examples, some of the components only achieve a folded state when the complex is formed. Striking results have appeared for systems ranging from the already mature field of virus structure and assembly, where notable progress has been made for rather complex capsids, to descriptions of ribosome structures in atomic detail, where recent results have emerged at breathtaking speed.

Butcher SJ, Makeyev EV, Grimes JM, Stuart DI, Bamford DH. 2000. Crystallization and preliminary X-ray crystallographic studies on the bacteriophage phi6 RNA-dependent RNA polymerase. Acta Crystallogr D Biol Crystallogr, 56 (Pt 11), pp. 1473-1475. | Show Abstract | Read more

The RNA-dependent RNA polymerase (P2) from bacteriophage Phi6 has been cloned and the protein overexpressed in Escherichia coli to produce an active enzyme. A fully substituted selenomethionyl version of the protein has also been produced. Crystals of both proteins have been grown; most belong to the monoclinic space group P2(1), with unit-cell parameters a = 105.9, b = 94.0, c = 140.9 A, beta = 101.4 degrees, but some are trigonal (space group P3(1) or P3(2)), with unit-cell parameters a = b = 110.1, c = 159.4 A, gamma = 120 degrees. Both crystal forms occur in the same crystallization drop and are morphologically indistinguishable. Native data sets have been collected from both types of crystals to better than 3 A resolution.

Ren J, Milton J, Weaver KL, Short SA, Stuart DI, Stammers DK. 2000. Structural basis for the resilience of efavirenz (DMP-266) to drug resistance mutations in HIV-1 reverse transcriptase. Structure, 8 (10), pp. 1089-1094. | Show Abstract | Read more

BACKGROUND: Efavirenz is a second-generation non-nucleoside inhibitor of HIV-1 reverse transcriptase (RT) that has recently been approved for use against HIV-1 infection. Compared with first-generation drugs such as nevirapine, efavirenz shows greater resilience to drug resistance mutations within HIV-1 RT. In order to understand the basis for this resilience at the molecular level and to help the design of further-improved anti-AIDS drugs, we have determined crystal structures of efavirenz and nevirapine with wild-type RT and the clinically important K103N mutant. RESULTS: The relatively compact efavirenz molecule binds, as expected, within the non-nucleoside inhibitor binding pocket of RT. There are significant rearrangements of the drug binding site within the mutant RT compared with the wild-type enzyme. These changes, which lead to the repositioning of the inhibitor, are not seen in the interaction with the first-generation drug nevirapine. CONCLUSIONS: The repositioning of efavirenz within the drug binding pocket of the mutant RT, together with conformational rearrangements in the protein, could represent a general mechanism whereby certain second-generation non-nucleoside inhibitors are able to reduce the effect of drug-resistance mutations on binding potency.

Maenaka K, Maenaka T, Tomiyama H, Takiguchi M, Stuart DI, Jones EY. 2000. Nonstandard peptide binding revealed by crystal structures of HLA-B*5101 complexed with HIV immunodominant epitopes. J Immunol, 165 (6), pp. 3260-3267. | Show Abstract

The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography. In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171. This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the alpha1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.

Mavaddat N, Mason DW, Atkinson PD, Evans EJ, Gilbert RJ, Stuart DI, Fennelly JA, Barclay AN, Davis SJ, Brown MH. 2000. Signaling lymphocytic activation molecule (CDw150) is homophilic but self-associates with very low affinity. J Biol Chem, 275 (36), pp. 28100-28109. | Show Abstract | Read more

Signaling lymphocytic activating molecule ((SLAM) CDw150) is a glycoprotein that belongs to the CD2 subset of the immunoglobulin superfamily and is expressed on the surface of activated T- and B-cells. It has been proposed that SLAM is homophilic and required for bidirectional signaling during T- and B-cell activation. Previous work has suggested that the affinity of SLAM self-association might be unusually high, undermining the concept that protein interactions mediating transient cell-cell contacts, such as those involving leukocytes, have to be weak in order that such contacts are readily reversible. Using surface plasmon resonance-based methods and analytical ultracentrifugation (AUC), we confirm that SLAM is homophilic. However, we also establish a new theoretical treatment of surface plasmon resonance-derived homophilic binding data, which indicates that SLAM-SLAM interactions (solution K(d) approximately 200 micrometer) are in fact considerably weaker than most other well characterized protein-protein interactions at the cell surface (solution K(d) approximately 0.4-20 micrometer), a conclusion that is supported by the AUC analysis. Whereas further analysis of the AUC data imply that SLAM could form "head to head" dimers spanning adjacent cells, the very low affinity raises important questions regarding the physiological role and/or properties of such interactions.

Gao GF, Willcox BE, Wyer JR, Boulter JM, O'Callaghan CA, Maenaka K, Stuart DI, Jones EY, Van Der Merwe PA, Bell JI, Jakobsen BK. 2000. Classical and nonclassical class I major histocompatibility complex molecules exhibit subtle conformational differences that affect binding to CD8alphaalpha. J Biol Chem, 275 (20), pp. 15232-15238. | Show Abstract | Read more

The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR). Here we use surface plasmon resonance to study the binding of CD8alphaalpha to class I MHC molecules. CD8alphaalpha bound the classical MHC molecules HLA-A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K(d)) of 90-220 microm, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8alphaalpha/classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8alphaalpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity (>/=1 mm), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8alphaalpha bound normally to the nonclassical MHC molecule HLA-G (K(d) approximately 150 microm), but only weakly to the natural killer cell receptor ligand HLA-E (K(d) >/= 1 mm). Site-directed mutagenesis experiments revealed that variation in CD8alphaalpha binding affinity can be explained by amino acid differences within the alpha3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.

Ren J, Nichols C, Bird LE, Fujiwara T, Sugimoto H, Stuart DI, Stammers DK. 2000. Binding of the second generation non-nucleoside inhibitor S-1153 to HIV-1 reverse transcriptase involves extensive main chain hydrogen bonding. J Biol Chem, 275 (19), pp. 14316-14320. | Show Abstract | Read more

S-1153 (AG1549) is perhaps the most promising non-nucleoside inhibitor of HIV-1 reverse transcriptase currently under development as a potential anti-AIDS drug, because it has a favorable profile of resilience to many drug resistance mutations. We have determined the crystal structure of S-1153 in a complex with HIV-1 reverse transcriptase. The complex possesses some novel features, including an extensive network of hydrogen bonds involving the main chain of residues 101, 103, and 236 of the p66 reverse transcriptase subunit. Such interactions are unlikely to be disrupted by side chain mutations. The reverse transcriptase/S-1153 complex suggests different ways in which resilience to mutations in the non-nucleoside inhibitors of reverse transcriptase binding site can be achieved.

Hogle JM, Stuart DI. 2000. Macromolecular assemblages - Editorial overview CURRENT OPINION IN STRUCTURAL BIOLOGY, 10 (2), pp. 205-207. | Read more

Ren J, Diprose J, Warren J, Esnouf RM, Bird LE, Ikemizu S, Slater M, Milton J, Balzarini J, Stuart DI, Stammers DK. 2000. Phenylethylthiazolylthiourea (PETT) non-nucleoside inhibitors of HIV-1 and HIV-2 reverse transcriptases. Structural and biochemical analyses. J Biol Chem, 275 (8), pp. 5633-5639. | Show Abstract | Read more

Most non-nucleoside reverse transcriptase (RT) inhibitors are specific for HIV-1 RT and demonstrate minimal inhibition of HIV-2 RT. However, we report that members of the phenylethylthiazolylthiourea (PETT) series of non-nucleoside reverse transcriptase inhibitors showing high potency against HIV-1 RT have varying abilities to inhibit HIV-2 RT. Thus, PETT-1 inhibits HIV-1 RT with an IC(50) of 6 nM but shows only weak inhibition of HIV-2 RT, whereas PETT-2 retains similar potency against HIV-1 RT (IC(50) of 5 nM) and also inhibits HIV-2 RT (IC(50) of 2.2 microM). X-ray crystallographic structure determinations of PETT-1 and PETT-2 in complexes with HIV-1 RT reveal the compounds bind in an overall similar conformation albeit with some differences in their interactions with the protein. To investigate whether PETT-2 could be acting at a different site on HIV-2 RT (e.g. the dNTP or template primer binding site), we compared modes of inhibition for PETT-2 against HIV-1 and HIV-2 RT. PETT-2 was a noncompetitive inhibitor with respect to the dGTP substrate for both HIV-1 and HIV-2 RTs. PETT-2 was also a noncompetitive inhibitor with respect to a poly(rC).(dG) template primer for HIV-2 RT. These results are consistent with PETT-2 binding in corresponding pockets in both HIV-1 and HIV-2 RT with amino acid sequence differences in HIV-2 RT affecting the binding of PETT-2 compared with PETT-1.

Ikemizu S, Gilbert RJ, Fennelly JA, Collins AV, Harlos K, Jones EY, Stuart DI, Davis SJ. 2000. Structure and dimerization of a soluble form of B7-1. Immunity, 12 (1), pp. 51-60. | Show Abstract | Read more

B7-1 (CD80) and B7-2 (CD86) are glycoproteins expressed on antigen-presenting cells. The binding of these molecules to the T cell homodimers CD28 and CTLA-4 (CD152) generates costimulatory and inhibitory signals in T cells, respectively. The crystal structure of the extracellular region of B7-1 (sB7-1), solved to 3 A resolution, consists of a novel combination of two Ig-like domains, one characteristic of adhesion molecules and the other previously seen only in antigen receptors. In the crystal lattice, sB7-1 unexpectedly forms parallel, 2-fold rotationally symmetric homodimers. Analytical ultracentrifugation reveals that sB7-1 also dimerizes in solution. The structural data suggest a mechanism whereby the avidity-enhanced binding of B7-1 and CTLA-4 homodimers, along with the relatively high affinity of these interactions, favors the formation of very stable inhibitory signaling complexes.

Robinson RC, Radziejewski C, Spraggon G, Greenwald J, Kostura MR, Burtnick LD, Stuart DI, Choe S, Jones EY. 1999. The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site. Protein Sci, 8 (12), pp. 2589-2597. | Show Abstract | Read more

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.

Hopkins AL, Ren J, Tanaka H, Baba M, Okamato M, Stuart DI, Stammers DK. 1999. Design of MKC-442 (emivirine) analogues with improved activity against drug-resistant HIV mutants. J Med Chem, 42 (22), pp. 4500-4505. | Show Abstract | Read more

Two analogues of the nonnucleoside inhibitor of HIV-1 RT, MKC-442 (emivirine), containing different C6 substituents have been designed to be less susceptible to the commonly found drug-resistance mutation of Tyr181Cys. Compound TNK-6123 had a C6 thiocyclohexyl group designed to have more flexibility in adapting to the mutated drug-binding site. GCA-186 had additional 3',5'-dimethyl substituents aimed at forming close contacts with the conserved residue Trp229. Both compounds showed approximately 30-fold greater inhibitory effect than MKC-442 to the Tyr181Cys mutant virus as well as to the clinically important Lys103Asn virus. X-ray crystallographic structure determination of complexes with HIV-1 RT confirmed the predicted binding modes. These strategies might be used to improve the resilience of other NNRTI series against common drug-resistance mutations.

Mongkolsapaya J, Grimes JM, Chen N, Xu XN, Stuart DI, Jones EY, Screaton GR. 1999. Structure of the TRAIL-DR5 complex reveals mechanisms conferring specificity in apoptotic initiation. Nat Struct Biol, 6 (11), pp. 1048-1053. | Show Abstract | Read more

TRAIL, an apoptosis inducing ligand, has at least four cell surface receptors including the death receptor DR5. Here we report the crystal structure at 2.2 A resolution of a complex between TRAIL and the extracellular region of DR5. TRAIL forms a central homotrimer around which three DR5 molecules bind. Radical differences in the surface charge of the ligand, together with variation in the alignment of the two receptor domains confer specificity between members of these ligand and receptor families. The existence of a switch mechanism allowing variation in receptor domain alignment may mean that it is possible to engineer receptors with multiple specificities by exploiting contact positions unique to individual receptor-ligand pairs.

Grimes JM, Fuller SD, Stuart DI. 1999. Complementing crystallography: the role of cryo-electron microscopy in structural biology. Acta Crystallogr D Biol Crystallogr, 55 (Pt 10), pp. 1742-1749. | Show Abstract | Read more

Dramatic improvements in experimental methods and computational techniques have revolutionized three-dimensional image reconstruction from electron micrographs (EM) of vitrified samples. Recent results include the first determination of a protein fold (for the core protein of the hepatitis B virus) by non-crystalline imaging techniques. These developments have generated interest within the crystallographic community and have led to a re-evaluation of the technique, particularly amongst those working in the field of virus structure or struggling with the phasing of large macromolecular assemblies. A simple discussion of the techniques of EM image reconstruction and its advantages and problems in terms familiar to crystallographers will hopefully allow an appreciation of the essential complementarity of the two techniques and the practical potentials for phasing applications.

Maenaka K, Juji T, Nakayama T, Wyer JR, Gao GF, Maenaka T, Zaccai NR, Kikuchi A, Yabe T, Tokunaga K et al. 1999. Killer cell immunoglobulin receptors and T cell receptors bind peptide-major histocompatibility complex class I with distinct thermodynamic and kinetic properties. J Biol Chem, 274 (40), pp. 28329-28334. | Show Abstract | Read more

Human natural killer cells and a subset of T cells express a repertoire of killer cell immunoglobulin receptors (KIRs) that recognize major histocompatibility complex (MHC) class I molecules. KIRs and T cell receptors (TCRs) bind in a peptide-dependent manner to overlapping regions of peptide-MHC class I complexes. KIRs with two immunoglobulin domains (KIR2Ds) recognize distinct subsets of HLA-C alleles. Here we use surface plasmon resonance to study the binding of soluble forms of KIR2DL1 and KIR2DL3 to several peptide-HLA-Cw7 complexes. KIR2DL3 bound to the HLA-Cw7 allele presenting the peptide RYRPGTVAL with a 1:1 stoichiometry and an affinity (K(d) approximately 7 microM at 25 degrees C) within the range of values measured for other cell-cell recognition molecules, including the TCR. Although KIR2DL1 is reported not to recognize the HLA-Cw7 allele in functional assays, it bound RYRPGTVAL/HLA-Cw7, albeit with a 10-20-fold lower affinity. TCR/peptide-MHC interactions are characterized by comparatively slow kinetics and unfavorable entropic changes (Willcox, B. E., Gao, G. F., Wyer, J. R. , Ladbury, J. E., Bell, J. I., Jakobsen, B. K., and van der Merwe, P. A. (1999) Immunity 10, 357-365), suggesting that binding is accompanied by conformational adjustments. In contrast, we show that KIR2DL3 binds RYRPGTVAL/HLA-Cw7 with fast kinetics and a favorable binding entropy, consistent with rigid body association. These results indicate that KIR/peptide-MHC class I interactions have properties typical of other cell-cell recognition molecules, and they highlight the unusual nature of TCR/peptide-MHC recognition.

Ren J, Esnouf RM, Hopkins AL, Stuart DI, Stammers DK. 1999. Crystallographic analysis of the binding modes of thiazoloisoindolinone non-nucleoside inhibitors to HIV-1 reverse transcriptase and comparison with modeling studies. J Med Chem, 42 (19), pp. 3845-3851. | Show Abstract | Read more

We have determined the crystal structures of thiazoloisoindolinone non-nucleoside inhibitors in complex with HIV-1 reverse transcriptase to high-resolution limits of 2.7 A (BM +21.1326) and 2. 52 A (BM +50.0934). We find that the binding modes of this series of inhibitors closely resemble that of "two-ring" non-nucleoside reverse transcriptase inhibitors. The structures allow rationalization of stereochemical requirements, structure-activity data, and drug resistance data. Comparisons with our previous structures suggest modifications to the inhibitors that might improve resilience to drug-resistant mutant forms of reverse transcriptase. Comparison with earlier modeling studies reveals that the predicted overlap of thiazoloisoindolinones with TIBO was largely correct, while that with nevirapine was significantly different.

Larder BA, Bloor S, Kemp SD, Hertogs K, Desmet RL, Miller V, Sturmer M, Staszewski S, Ren J, Stammers DK et al. 1999. A family of insertion mutations between codons 67 and 70 of human immunodeficiency virus type 1 reverse transcriptase confer multinucleoside analog resistance. Antimicrob Agents Chemother, 43 (8), pp. 1961-1967. | Show Abstract

To investigate the occurrence of multinucleoside analog resistance during therapy failure, we surveyed the drug susceptibilities and genotypes of nearly 900 human immunodeficiency virus type 1 (HIV-1) samples. For 302 of these, the 50% inhibitory concentrations of at least four of the approved nucleoside analogs had fourfold-or-greater increases. Genotypic analysis of the reverse transcriptase (RT)-coding regions from these samples revealed complex mutational patterns, including the previously recognized codon 151 multidrug resistance cluster. Surprisingly, high-level multinucleoside resistance was associated with a diverse family of amino acid insertions in addition to "conventional" point mutations. These insertions were found between RT codons 67 and 70 and were commonly 69Ser-(Ser-Ser) or 69Ser-(Ser-Gly). Treatment history information showed that a common factor for the development of these variants was AZT (3'-azido-3'-deoxythymidine, zidovudine) therapy in combination with 2',3'-dideoxyinosine or 2',3'-dideoxycytidine, although treatment patterns varied considerably. Site-directed mutagenesis studies confirmed that 69Ser-(Ser-Ser) in an AZT resistance mutational background conferred simultaneous resistance to multiple nucleoside analogs. The insertions are located in the "fingers" domain of RT. Modelling the 69Ser-(Ser-Ser) insertion into the RT structure demonstrated the profound direct effect that this change is likely to have in the nucleoside triphosphate binding site of the enzyme. Our data highlight the increasing problem of HIV-1 multidrug resistance and underline the importance of continued resistance surveillance with appropriate, sufficiently versatile genotyping technology and phenotypic drug susceptibility analysis.

Verdaguer N, Marlovits TC, Bravo J, Stuart DI, Blaas D, Fita I. 1999. Crystallization and preliminary X-ray analysis of human rhinovirus serotype 2 (HRV2). Acta Crystallogr D Biol Crystallogr, 55 (Pt 8), pp. 1459-1461. | Show Abstract | Read more

Human rhinoviruses, the major cause of mild recurrent infections of the upper respiratory tract, are small icosahedral particles. Over 100 different serotypes have been identified. The majority (91 serotypes) use intercellular adhesion molecule 1 as the cell-attachment site; ten serotypes (the minor group) bind to members of the low-density lipoprotein receptor. Three different crystal forms of the minor-group human rhinovirus serotype 2 (HRV2) were obtained by the hanging-drop vapour-diffusion technique using ammonium sulfate and sodium/potassium phosphate as precipitants. Monoclinic crystals, space group P2(1), diffracted at least to 2.8 A resolution, and two complete virus particles were located in the crystal asymmetric unit. A second type of crystals had a compact cubic like morphology and diffracted beyond 2.5 A resolution. These crystals belong to a primitive orthorhombic space group, with unit-cell parameters a = 309.3, b = 353.5, c = 759.6 A, and contain one virus particle in the asymmetric unit. A third type of crystals, with a prismatic shape and belonging to space group I222, was also obtained under similar crystallization conditions. These latter crystals, with unit-cell parameters a = 308.7, b = 352.2, c = 380.5 A, diffracted to high resolution (beyond 1.8 A) and contained 15 protomers per asymmetric unit; this requires that three perpendicular crystal twofold axes coincide with three of the viral particle's dyad axes.

Butters TD, Sparks LM, Harlos K, Ikemizu S, Stuart DI, Jones EY, Davis SJ. 1999. Effects of N-butyldeoxynojirimycin and the Lec3.2.8.1 mutant phenotype on N-glycan processing in Chinese hamster ovary cells: application to glycoprotein crystallization. Protein Sci, 8 (8), pp. 1696-1701. | Show Abstract | Read more

Heterologous gene expression in either (1) the glycosylation-defective, mutant Chinese hamster ovary cell line, Lec3.2.8.1, or (2) the presence of the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin facilitates the trimming of N-linked glycans of glycoproteins to single N-acetylglucosamine (GlcNAc) residues with endoglycosidase H (endo H). Both approaches are somewhat inefficient, however, with as little as 12% of the total protein being rendered fully endo H-sensitive under these conditions. It is shown here that the combined effects of these approaches on the restriction of oligosaccharide processing are essentially additive, thereby allowing the production of glycoproteins that are essentially completely endo H-sensitive. The preparation of a soluble chimeric form of CD58, the ligand of the human T-cell surface recognition molecule CD2, illustrates the usefulness of the combined approach when expression levels are low or the deglycosylated protein is unstable at low pH. The endo H-treated chimera produced crystals of space group P3(1)21 or P3(2)21, and unit cell dimensions a = b = 116.4 A, c = 51.4 A alpha = beta = 90 degrees , gamma = 120 degrees , that diffract to a maximum resolution of 1.8 A.

Ellard FM, Drew J, Blakemore WE, Stuart DI, King AM. 1999. Evidence for the role of His-142 of protein 1C in the acid-induced disassembly of foot-and-mouth disease virus capsids. J Gen Virol, 80 ( Pt 8) (8), pp. 1911-1918. | Show Abstract | Read more

Foot-and-mouth disease virus (FMDV) capsids are inherently labile under mildly acidic conditions, dissociating to pentamers at pH values in the region of 6.5, with the release of protein 1A and the viral RNA. This acid-induced disassembly is thought to be required for the entry of the virus genome into the host cell. Previous work has highlighted a histidine-alpha-helix charge-dipole interaction at the twofold axes of symmetry between pentamers and has suggested that this interaction plays a role in acid-induced disassembly. The validity of this theory has now been tested by converting the implicated residue, His-142 of protein 1C, to Arg, Phe and Asp. The effects of such changes were studied by using a previously described vaccinia virus expression system, in which synthesis and processing of FMDV capsid proteins results in the self-assembly of capsids. In agreement with the histidine-alpha-helix charge-dipole theory, assembly in the arginine mutant was found to be greatly reduced, while capsids of the aspartic acid mutant were considerably more stable under acidic conditions than the wild-type. Aberrant but acid-stable complexes were obtained in the phenylalanine mutant.

Fry EE, Grimes J, Stuart DI. 1999. Virus crystallography. Mol Biotechnol, 12 (1), pp. 13-23. | Show Abstract | Read more

Virus crystallography can provide atomic resolution structures for intact isometric virus particles and components thereof. The methodology is illustrated by reference to a particularly complex example, the core of the bluetongue virus (700 A).

Diprose JM, Grimes JM, Gouet P, Malby R, Burroughs JN, Lescar J, Rassmussen B, Mertens PPC, Stuart DI. 1999. Bluetongue virus: the role of synchrotron radiation JOURNAL OF SYNCHROTRON RADIATION, 6 (4), pp. 865-874. | Show Abstract | Read more

The determination of the structure of the transcriptionally active core particle of bluetongue virus is discussed. This particle is approximately 700 Å in diameter and reasonably well ordered, but fragile, crystals have been obtained from two different serotypes of the virus. Cryocrystallography proved difficult and a large number of crystals were analysed at room temperature to accumulate a reasonably complete data set. The effects of synchrotron optics, station design and detector on the signal-to-noise for these weak data are discussed, with particular reference to station ID2 at the European Synchrotron Radiation Facility. Once the data had been gathered, structure determination was straightforward, using a model derived from a combination of electron microscopy and protein crystallography to obtain initial phases. Despite apparent isomorphism, it is suspected that the crystal lattice 'ages', perhaps reflecting both the inevitable weakness of the forces holding crystals of such a large macromolecular complex together and flexibility in the particle.

Hill CL, Booth TF, Prasad BV, Grimes JM, Mertens PP, Sutton GC, Stuart DI. 1999. The structure of a cypovirus and the functional organization of dsRNA viruses. Nat Struct Biol, 6 (6), pp. 565-568. | Show Abstract | Read more

Cytoplasmic polyhedrosis virus (CPV) is unique among the double-stranded RNA viruses of the family Reoviridae in having a single capsid layer. Analysis by cryo-electron microscopy allows comparison of the single shelled CPV and orthoreovirus with the high resolution crystal structure of the inner shell of the bluetongue virus (BTV) core. This suggests that the novel arrangement identified in BTV, of 120 protein subunits in a so-called 'T=2' organization, is a characteristic of the Reoviridae and allows us to delineate structural similarities and differences between two subgroups of the family--the turreted and the smooth-core viruses. This in turn suggests a coherent picture of the structural organization of many dsRNA viruses.

Gouet P, Diprose JM, Grimes JM, Malby R, Burroughs JN, Zientara S, Stuart DI, Mertens PP. 1999. The highly ordered double-stranded RNA genome of bluetongue virus revealed by crystallography. Cell, 97 (4), pp. 481-490. | Show Abstract | Read more

The concentration of double-stranded RNA within the bluetongue virus core renders the genome segments liquid crystalline. Powder diffraction rings confirm this local ordering with a 30 A separation between strands. Determination of the structure of the bluetongue virus core serotype 10 and comparison with that of serotype 1 reveals most of the genomic double-stranded RNA, packaged as well-ordered layers surrounding putative transcription complexes at the apices of the particle. The outer layer of RNA is sufficiently well ordered by interaction with the capsid that a model can be built and extended to the less-ordered inner layers, providing a structural framework for understanding the mechanism of this complex transcriptional machine. We show that the genome segments maintain local order during transcription.

Paesen GC, Adams PL, Harlos K, Nuttall PA, Stuart DI. 1999. Tick histamine-binding proteins: isolation, cloning, and three-dimensional structure. Mol Cell, 3 (5), pp. 661-671. | Show Abstract | Read more

High-affinity histamine-binding proteins (HBPs) were discovered in the saliva of Rhipicephalus appendiculatus ticks. Their ability to outcompete histamine receptors indicates that they suppress inflammation during blood feeding. The crystal structure of a histamine-bound HBP, determined at 1.25 A resolution, reveals a lipocalin fold novel in containing two binding sites for the same ligand. The sites are orthogonally arranged and highly rigid and form an internal surface of unusual polar character that complements the physicochemical properties of histamine. As soluble receptors of histamine, HBPs offer a new strategy for controlling histamine-based diseases.

Maenaka K, Juji T, Stuart DI, Jones EY. 1999. Crystal structure of the human p58 killer cell inhibitory receptor (KIR2DL3) specific for HLA-Cw3-related MHC class I. Structure, 7 (4), pp. 391-398. | Show Abstract | Read more

BACKGROUND: T cells and natural killer (NK) cells perform complementary roles in the cellular immune system. T cells identify infected cells directly through recognition of antigenic peptides that are displayed at the target cell surface by the classical major histocompatibility complex (MHC) class I molecules. NK cells monitor the target cell surface for malfunction of this display system, lysing potentially infected cells that might otherwise evade recognition by the T cells. Human killer cell inhibitory receptors (KIRs) control this process by either inhibiting or activating the cytotoxic activity of NK cells via specific binding to MHC class I molecules on the target cell. RESULTS: We report the crystal structure of the extracellular region of the human p58 KIR (KIR2DL3), which is specific for the human MHC class I molecule HLA-Cw3 and related alleles. The structure shows the predicted topology of two tandem immunoglobulin-like domains, but comparison with the previously reported structure of the related receptor KIR2DL1 reveals an unexpected change of 23 degrees in the relative orientation of these domains. CONCLUSIONS: The altered orientation of the immunoglobulin-like domains maintains an unusually acute interdomain elbow angle, which therefore appears to be a distinctive feature of the KIRs. The putative MHC class I binding site is located on the outer surface of the elbow, spanning both domains. The unexpected observation that this binding site can be modulated by differences in the relative domain orientations has implications for the general mechanism of KIR-MHC class I complex formation.

Ikemizu S, Sparks LM, van der Merwe PA, Harlos K, Stuart DI, Jones EY, Davis SJ. 1999. Crystal structure of the CD2-binding domain of CD58 (lymphocyte function-associated antigen 3) at 1.8-A resolution. Proc Natl Acad Sci U S A, 96 (8), pp. 4289-4294. | Show Abstract | Read more

The binding of the cell surface molecule CD58 (formerly lymphocyte function-associated antigen 3) to its ligand, CD2, significantly increases the sensitivity of antigen recognition by T cells. This was the first heterophilic cell adhesion interaction to be discovered and is now an important paradigm for analyzing the structural basis of cell-cell recognition. The crystal structure of a CD2-binding chimeric form of CD58, solved to 1.8-A resolution, reveals that the ligand binding domain of CD58 has the expected Ig superfamily V-set topology and shares several of the hitherto unique structural features of CD2, consistent with previous speculation that the genes encoding these molecules arose via duplication of a common precursor. Nevertheless, evidence for considerable divergence of CD2 and CD58 is also implicit in the structures. Mutations that disrupt CD2 binding map to the highly acidic surface of the AGFCC'C" beta-sheet of CD58, which, unexpectedly, lacks marked shape complementarity to the equivalent, rather more basic CD58-binding face of human CD2. The specificity of the very weak interactions of proteins mediating cell-cell recognition may often derive largely from electrostatic complementarity, with shape matching at the protein-protein interface being less exact than for interactions that combine specificity with high affinity, such as those involving antibodies.

Gouet P, Courcelle E, Stuart DI, Métoz F. 1999. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics, 15 (4), pp. 305-308. | Show Abstract | Read more

MOTIVATION: The program ESPript (Easy Sequencing in PostScript) allows the rapid visualization, via PostScript output, of sequences aligned with popular programs such as CLUSTAL-W or GCG PILEUP. It can read secondary structure files (such as that created by the program DSSP) to produce a synthesis of both sequence and structural information. RESULTS: ESPript can be run via a command file or a friendly html-based user interface. The program calculates an homology score by columns of residues and can sort this calculation by groups of sequences. It offers a palette of markers to highlight important regions in the alignment. ESPript can also paste information on residue conservation into coordinate files, for subsequent visualization with a graphics program. AVAILABILITY: ESPript can be accessed on its Web site at http://www.ipbs.fr/ESPript. Sources and helpfiles can be downloaded via anonymous ftp from ftp.ipbs.fr. A tar file is held in the directory pub/ESPript.

Sharma A, Askari JA, Humphries MJ, Jones EY, Stuart DI. 1999. Crystal structure of a heparin- and integrin-binding segment of human fibronectin. EMBO J, 18 (6), pp. 1468-1479. | Show Abstract | Read more

The crystal structure of human fibronectin (FN) type III repeats 12-14 reveals the primary heparin-binding site, a clump of positively charged residues in FN13, and a putative minor site approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 binding is at the FN13-14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for alpha4beta1 binding to FN12-14. The PRARI motif may represent a new class of integrin-binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert.

Hill CL, Booth TF, Stuart DI, Mertens PP. 1999. Lipofectin increases the specific activity of cypovirus particles for cultured insect cells. J Virol Methods, 78 (1-2), pp. 177-189. | Show Abstract | Read more

Cytoplasmic polyhedrosis viruses (CPV) are classified as 14 distinct species (electropherotypes) within the genus Cypovirus, family Reoviridae. Cypovirus research has been limited by a lack of appropriate cell culture systems (for each of these virus species) in which the majority of cells can become productively infected. Lipofection increased the infection rate of Lymantria dispar 652 cells, by virus particles (derived from polyhedra) of Orgyia pseudosugata type 5 cypovirus (Op-5 CPV), from 3 to 44%. Lipofection also significantly increased the percentage of Trichoplusia ni 368 cells infected with the same virus (from < 1 to approximately 7%). The spread of cypovirus infection between cells was either very slow or insignificant, and infected cells appeared to remain viable for long periods. Virus infection was detected by the observation of polyhedra formation in individual cells and it was therefore possible to develop a simple quantitative assay system to measure virus titre (TCID50). Cryo-electron microscopy showed that cypovirus particles formed a complex with the lipid, involving their envelopment within the liposome membrane. It was concluded that the increased infectivity of the virus by lipofection was due to a more efficient cell entry mechanism, probably involving fusion between liposome and cell membranes.

Fry EE, Lea SM, Jackson T, Newman JW, Ellard FM, Blakemore WE, Abu-Ghazaleh R, Samuel A, King AM, Stuart DI. 1999. The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex. EMBO J, 18 (3), pp. 543-554. | Show Abstract | Read more

Heparan sulfate has an important role in cell entry by foot-and-mouth disease virus (FMDV). We find that subtype O1 FMDV binds this glycosaminoglycan with a high affinity by immobilizing a specific highly abundant motif of sulfated sugars. The binding site is a shallow depression on the virion surface, located at the junction of the three major capsid proteins, VP1, VP2 and VP3. Two pre-formed sulfate-binding sites control receptor specificity. Residue 56 of VP3, an arginine in this virus, is critical to this recognition, forming a key component of both sites. This residue is a histidine in field isolates of the virus, switching to an arginine in adaptation to tissue culture, forming the high affinity heparan sulfate-binding site. We postulate that this site is a conserved feature of FMDVs, such that in the infected animal there is a biological advantage to low affinity, or more selective, interactions with glycosaminoglycan receptors.

Lea SM, Powell RM, McKee T, Evans DJ, Brown D, Stuart DI, van der Merwe PA. 1998. Determination of the affinity and kinetic constants for the interaction between the human virus echovirus 11 and its cellular receptor, CD55. J Biol Chem, 273 (46), pp. 30443-30447. | Show Abstract | Read more

The biochemical properties of the molecular interactions mediating viral-cell recognition are poorly characterized. In this study, we use surface plasmon resonance to study the affinity and kinetics of the interaction of echovirus 11 with its cellular receptor decay-accelerating factor (CD55). As reported for interactions between cell-cell recognition molecules, the interaction has a low affinity (KD approximately 3.0 microM) as a result of a very fast dissociation rate constant (kon approximately 10(5) M-1.s-1, koff approximately 0.3 s-1). This contrasts with the interaction of soluble ICAM-1 (sICAM-1, CD54) with human rhinovirus 3 which has been reported to have a similar affinity but 10(2)-10(3)-fold slower kinetics (Casasnovas, J. M., and Springer, T. A. (1995) J. Biol. Chem. 270, 13216-13224). The extracellular portion of decay-accelerating factor comprises four short consensus repeat domains (domains 1-4) and a mucin-like stalk. By comparison of the binding affinity for echovirus 11 of various fragments of decay-accelerating factor, we are able to conclude that short consensus repeat domain 3 contributes approximately 80% of the binding energy.

Ren J, Esnouf RM, Hopkins AL, Warren J, Balzarini J, Stuart DI, Stammers DK. 1998. Crystal structures of HIV-1 reverse transcriptase in complex with carboxanilide derivatives. Biochemistry, 37 (41), pp. 14394-14403. | Show Abstract | Read more

The carboxanilides are nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT), of potential clinical importance. The compounds differ in potency and in their retention of potency in the face of drug resistance mutations. Whereas UC-84, the prototype compound, only weakly inhibits many RTs bearing single point resistance mutations, inhibition by UC-781 is little affected. It has been proposed that UC-38 and UC-781 may form quaternary complexes with RT at a site other than the known binding pocket of other NNIs. X-ray crystal structures of four HIV-1 RT-carboxanilide complexes (UC-10, UC-38, UC-84, and UC-781) reported here reveal that all four inhibitors bind in the usual NNI site, forming binary 1:1 complexes with RT in the absence of substrates with the amide/thioamide bond in cis conformations. For all four complexes the anilide rings of the inhibitors overlap aromatic rings of many other NNIs bound to RT. In contrast, the second rings of UC-10, UC-84, and UC-781 do not bind in equivalent positions to those of other "two-ring" NNIs such as alpha-APA or HEPT derivatives. The binding modes most closely resemble that of the structurally dissimilar NNI, Cl-TIBO, with a common hydrogen bond between each carboxanilide NH- group and the main-chain carbonyl oxygen of Lys101. The binding modes differ slightly between the UC-10/UC-781 and UC-38/UC-84 pairs of compounds, apparently related to the shorter isopropylmethanoyl substituents of the anilide rings of UC-38/UC-84, which draws these rings closer to residues Tyr181 and Tyr188. This in turn explains the differences in the effect of mutated residues on the binding of these compounds.

Grimes JM, Burroughs JN, Gouet P, Diprose JM, Malby R, Ziéntara S, Mertens PP, Stuart DI. 1998. The atomic structure of the bluetongue virus core. Nature, 395 (6701), pp. 470-478. | Show Abstract | Read more

The structure of the core particle of bluetongue virus has been determined by X-ray crystallography at a resolution approaching 3.5 A. This transcriptionally active compartment, 700 A in diameter, represents the largest molecular structure determined in such detail. The atomic structure indicates how approximately 1,000 protein components self-assemble, using both the classical mechanism of quasi-equivalent contacts, which are achieved through triangulation, and a different method, which we term geometrical quasi-equivalence.

Esnouf RM, Ren J, Garman EF, Somers DO, Ross CK, Jones EY, Stammers DK, Stuart DI. 1998. Continuous and discontinuous changes in the unit cell of HIV-1 reverse transcriptase crystals on dehydration. Acta Crystallogr D Biol Crystallogr, 54 (Pt 5), pp. 938-953. | Show Abstract | Read more

A crystal form of HIV-1 reverse transcriptase (RT) complexed with inhibitors showed diffraction to a high-resolution limit of 3.7 A. Instability in the unit-cell dimensions of these crystals was observed during soaking experiments, but the range of this variability and consequent change in lattice order was revealed by a chance observation of dehydration. Deliberately induced dehydration results in crystals having a variety of unit cells, the best-ordered of which show diffraction to a minimum Bragg spacing of 2.2 A. In order to understand the molecular basis for this phenomenon, the initial observation of dehydration, the data sets from dehydrated crystals, the crystal packing and the domain conformation of RT are analysed in detail here. This analysis reveals that the crystals undergo remarkable changes following a variety of possible dehydration pathways: some changes occur gradually whilst others are abrupt and require significant domain rearrangements. Comparison of domain arrangements in different crystal forms gives insight into the flexibility of RT which, in turn, may reflect the internal motions allowing this therapeutically important enzyme to fulfill its biological function.

Ren J, Esnouf RM, Hopkins AL, Jones EY, Kirby I, Keeling J, Ross CK, Larder BA, Stuart DI, Stammers DK. 1998. 3'-Azido-3'-deoxythymidine drug resistance mutations in HIV-1 reverse transcriptase can induce long range conformational changes. Proc Natl Acad Sci U S A, 95 (16), pp. 9518-9523. | Show Abstract | Read more

HIV reverse transcriptase (RT) is one of the main targets for the action of anti-AIDS drugs. Many of these drugs [e.g., 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI)] are analogues of the nucleoside substrates used by the HIV RT. One of the main problems in anti-HIV therapy is the selection of a mutant virus with reduced drug sensitivity. Drug resistance in HIV is generated for nucleoside analogue inhibitors by mutations in HIV RT. However, most of these mutations are situated some distance from the polymerase active site, giving rise to questions concerning the mechanism of resistance. To understand the possible structural bases for this, the crystal structures of AZT- and ddI-resistant RTs have been determined. For the ddI-resistant RT with a mutation at residue 74, no significant conformational changes were observed for the p66 subunit. In contrast, for the AZT-resistant RT (RTMC) bearing four mutations, two of these (at 215 and 219) give rise to a conformational change that propagates to the active site aspartate residues. Thus, these drug resistance mutations produce an effect at the RT polymerase site mediated simply by the protein. It is likely that such long-range effects could represent a common mechanism for generating drug resistance in other systems.

Grimes JM, Stuart DI. 1998. Large unit cells and cellular mechanics. Nat Struct Biol, 5 Suppl (8), pp. 630-634. | Show Abstract | Read more

Developments in synchrotron radiation mean that the methodological and technological tools are in place to determine the structures of large multi- component macromolecular machines.

Tanaka H, Walker RT, Hopkins AL, Ren J, Jones EY, Fujimoto K, Hayashi M, Miyasaka T, Baba M, Stammers DK, Stuart DI. 1998. Allosteric inhibitors against HIV-1 reverse transcriptase: design and synthesis of MKC-442 analogues having an omega-functionalized acyclic structure. Antivir Chem Chemother, 9 (4), pp. 325-332. | Show Abstract

Based on X-ray crystallographic analysis of MKC-442/human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) complex, analogues in which the N1-substituent is replaced with omega-functionalized alkyl groups were designed to improve the affinity for the enzyme. Synthesis of these compounds was carried out starting from MKC-442 by a sequence of reactions (N3-protection, removal of N1-ethoxymethyl group, alkylation, and N3-deprotection). The compounds were evaluated for anti-HIV activity. Structure-activity relationships are discussed in terms of the possible interaction with the enzyme.

Jones EY, Tormo J, Reid SW, Stuart DI. 1998. Recognition surfaces of MHC class I. Immunol Rev, 163 (1), pp. 121-128. | Show Abstract | Read more

Recent crystallographic results have provided close to atomic resolution views of the recognition events mediated by MHC class I molecules. The specificity-conferring interaction of MHC class I/peptide with a T-cell antigen receptor (TCR) appears dependent on certain key interactions with the MHC scaffold. These interactions, in particular those of the TCR V alpha domain, define a standard orientation for TCR binding. Previous studies on biologically significant variations in the TCR recognition surface presented by a series of MHC/variant peptide complexes can be reassessed in the light of this TCR-binding mode. The interaction of CD8 with MHC class I resembles that between antibody and antigen in the use of loops from the CD8 structure. The interaction is of very low affinity and buries equivalent surface area to that between the TCR and MHC class I but while the TCR/MHC interface shows poor surface shape complementarity the match in the conservative interaction between MHC and CD8 is precise.

Maenaka K, Juji T, Tadokoro K, Harlos K, Stuart DI, Jones EY. 1998. Crystallization and preliminary diffraction studies of the extracellular region of human p58 killer cell inhibitory receptor (KIR2). Acta Crystallogr D Biol Crystallogr, 54 (Pt 3), pp. 433-435. | Show Abstract | Read more

Molecules of the human killer cell inhibitory receptor (KIR) family, which belong to the immunoglobulin superfamily (IgSF), are expressed on the surface of natural killer (NK) cells and some subsets of T cells. These receptors function to mediate the inhibition or activation of cytotoxic activity by recognizing HLA class I molecules on the target cell. The extracellular region of a p58 KIR specific for HLA-Cw1,3,7 (KIR2) has been overproduced in Escherichia coli and purified. The recombinant KIR2 has been crystallized in 9-10% poly(ethylene glycol) methyl ether (average Mr = 8000), 50mM HEPES, 8% ethylene glycol, 0.5% octyl-beta-glucoside, pH 7.5, at 294 K using the sitting-drop vapour-diffusion method. Preliminary X-ray diffraction studies reveal the space group to be hexagonal (P6122 or P6522) with lattice constants a = b = 95.3, c = 130.8 A. A native data set (3 A resolution) has been collected at the Photon Factory (lambda = 1.0 A).

O'Callaghan CA, Tormo J, Willcox BE, Blundell CD, Jakobsen BK, Stuart DI, McMichael AJ, Bell JI, Jones EY. 1998. Production, crystallization, and preliminary X-ray analysis of the human MHC class Ib molecule HLA-E. Protein Sci, 7 (5), pp. 1264-1266. | Show Abstract | Read more

HLA-E is the first human class Ib major histocompatibility complex molecule to be crystallized. HLA-E is highly conserved and almost nonpolymorphic, and has recently been shown to be the first specialized ligand for natural killer cell receptors. In functional studies, HLA-E is unlike the class Ia MHC molecules in having tightly restricted peptide binding specificity. HLA-E binds a limited set of almost identical leader sequence peptides derived from class Ia molecules and presents these at the cell surface for recognition by natural killer cell receptors. We now show that the extracellular region of HLA-E forms a stable complex with beta2 microglobulin and can be refolded around synthetic peptide. Crystals of this complex formed slowly over four to six months in the presence of ammonium sulphate. The crystals diffract to 2.85 A with space group P3(1)21 and unit cell dimensions a = 182.2 A, b = 182.2 A, c = 88.4 A.

Paesen GC, Adams P, Harlos K, Stuart DI, Nuttall PA. 1998. Histamine - Binding proteins in tick saliva FASEB JOURNAL, 12 (5), pp. A1001-A1001. | Show Abstract

Ticks, like other haematophagous ectoparasites, secrete in their saliva bioactive substances that counter the host animal's defence reactions at the feeding site and thus facilitate the acquisition of the bloodmeal. We discovered a family of histamine-binding proteins (HBPs) in the saliva of the brown ear tick, Rhipicephalus appendiculatus. HBPs most probably act by mopping up free histamine at the wound site, making it unavailable to the host's H1- and H2-histamine receptors, thus suppressing inflammation. The tick proteins show no sequence homology with known histamine receptors, nor do histamine receptor agonists or antagonists effectively compete with histamine for the binding sites on the tick proteins. The HBPs were expressed in a baculovirus-insect cell expression system. Binding-assays with 3H-histamine reveal high affinities for two of the proteins, but a weaker binding for a third HBP. The high-affinity binders very efficiently suppress histamine-induced contraction of guinea pig ileum. One of the HBPs was crystallized in the presence of histamine, and its structure determined at a 1.2 angstrom resolution. The protein appears to be a member of the lipocalin family, and contains both a high and low affinity histamine-binding site.

O'Callaghan CA, Tormo J, Willcox BE, Braud VM, Jakobsen BK, Stuart DI, McMichael AJ, Bell JI, Jones EY. 1998. Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E. Mol Cell, 1 (4), pp. 531-541. | Show Abstract | Read more

The crystal structure of the nonclassical human class lb MHC molecule HLA-E has been determined in complex with a prototypic ligand, the nonamer peptide (VMAPRTVLL), derived from the highly conserved residues 3-11 of the human MHC class la leader sequence. The mode of peptide binding retains some of the standard features observed in MHC class la complexes, but novel features imply that HLA-E has evolved to mediate specific binding to a tightly defined set of almost identical hydrophobic peptides from the highly conserved class l leader sequences. These molecular adaptations make HLA-E a rigorous checkpoint at the cell surface reporting on the integrity of the antigen processing pathway to CD94/NKG2 receptor-bearing natural killer cells.

Sharma A, Rao Z, Fry E, Booth T, Jones EY, Rowlands DJ, Simmons DL, Stuart DI. 1997. Specific interactions between human integrin alpha v beta 3 and chimeric hepatitis B virus core particles bearing the receptor-binding epitope of foot-and-mouth disease virus. Virology, 239 (1), pp. 150-157. | Show Abstract | Read more

Purified integrin alpha v beta 3 was used in solid-phase binding studies with chimeric hepatitis B cores which carry the RGD-containing loop of VP1 protein of the foot-and-mouth disease virus (FMDV). High levels of specific binding between the integrin and the particles were detected by enzyme-linked immunosorbent assays. The binding was Mn2+ cation dependent and could be competed with fibronectin, vitronectin, and the peptide GRGDSPK. Particles in which the RGD motif had been mutated to RGE failed to bind, indicating that the chimeric cores bound specifically to the ligand binding site of integrin alpha v beta 3. Electron micrographs showed several individual alpha v beta 3 molecules bound to the surface of each chimeric particle. Collectively, these data constitute firm evidence that the RGD-containing loop of FMDV is critical for binding to alpha v beta 3 and provide support for identification of alpha v beta 3 as a potential cellular receptor for FMDV.

Jackson T, Sharma A, Ghazaleh RA, Blakemore WE, Ellard FM, Simmons DL, Newman JW, Stuart DI, King AM. 1997. Arginine-glycine-aspartic acid-specific binding by foot-and-mouth disease viruses to the purified integrin alpha(v)beta3 in vitro. J Virol, 71 (11), pp. 8357-8361. | Show Abstract

The integrin alpha(v)beta3 has been shown to act as the receptor for internalization of foot-and-mouth disease virus (FMDV) (A12), with attachment being through a highly conserved RGD motif located on the G-H loop of viral capsid protein VP1. In addition, however, we have recently shown that efficient infection of culture-grown cells by FMDV (O1BFS) requires binding to cell surface heparan sulfate. In this study, we have used a solid-phase receptor binding assay to characterize the binding by FMDV to purified alpha(v)beta3 in the absence of heparan sulfate and other cell surface components. In this assay, FMDV (O1BFS) successfully replicated authentic ligand binding by cellular alpha(v)beta3 in terms of its high affinity, dependence on divalent cations, and activation by manganese ions. Virus binding to this preparation of alpha(v)beta3 was exquisitely sensitive to competition by short RGD-containing peptides (50% inhibition at < 10(-8) M peptide), and this inhibition was highly sequence specific, with the equivalent RGE peptide being at least 10(4) fold less effective as a competitor. Representative viruses of the other six serotypes of FMDV bound to alpha(v)beta3 in a similar RGD-specific manner, although significant differences in sensitivity to RGD peptides suggest that the affinity of the different FMDV serotypes for alpha(v)beta3 is influenced, in part, by the variable amino acid residues in the VP1 G-H loop on either side of the RGD.

Burtnick LD, Koepf EK, Grimes J, Jones EY, Stuart DI, McLaughlin PJ, Robinson RC. 1997. The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation. Cell, 90 (4), pp. 661-670. | Show Abstract | Read more

The structure of gelsolin has been determined by crystallography and comprises six structurally related domains that, in a Ca2+-free environment, pack together to form a compact globular structure in which the putative actin-binding sequences are not sufficiently exposed to enable binding to occur. We propose that binding Ca2+ can release the connections that join the N- and C-terminal halves of gelsolin, enabling each half to bind actin relatively independently. Domain shifts are proposed in response to Ca2+ as bases for models of how gelsolin acts to sever, cap, or nucleate F-actin filaments. The structure also invites discussion of polyphosphoinositide binding to segment 2 and suggests how mutation at Asp-187 could initiate a series of events that lead to deposition of amyloid plaques, as observed in victims of familial amyloidosis (Finnish type).

Grimes JM, Jakana J, Ghosh M, Basak AK, Roy P, Chiu W, Stuart DI, Prasad BV. 1997. An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy. Structure, 5 (7), pp. 885-893. | Show Abstract | Read more

BACKGROUND: Bluetongue virus (BTV), which belongs to the Reoviridae family and orbivirus genus, is a non-enveloped, icosahedral, double-stranded RNA virus. Several protein layers enclose its genome; upon cell entry the outer layer is stripped away leaving a core, the surface of which is composed of VP7. The structure of the trimeric VP7 molecule has previously been determined using X-ray crystallography. The articulated VP7 subunit consists of two domains, one which is largely alpha-helical and the other, smaller domain, is a beta barrel with jelly-roll topology. The relative orientations of these two domains vary in different crystal forms. The structure of VP7 and the organizations of 780 subunits of this molecule in the core of virus is central to the assembly and function of BTV. RESULTS: A 23 A resolution map of the core, determined using electron cryomicroscopy (cryoEM) data, reveals that the 260 trimers of VP7 are organized on a rather precise T = 13 laevo icosahedral lattice, in accordance with the theory of quasi-equivalence. The VP7 layer occupies a shell that is between 260 A and 345 A from the centre of the core. Below this radius (230-260 A) lies the T = 1 layer of 120 molecules of VP3. By fitting the X-ray structure of an individual VP7 trimer onto the cryoEM BTV core structure, we have generated an atomic model of the VP7 layer of BTV. This demonstrates that one of the molecular structures seen in crystals of the isolated VP7 corresponds to the in vivo conformation of the molecule in the core. CONCLUSIONS: The beta-barrel domains of VP7 are external to the core and interact with protein in the outer layer of the mature virion. The lower, alpha-helical domains of VP7 interact with VP3 molecules which form the inner layer of the BTV core. Adjacent VP7 trimer-trimer interactions in the T = 13 layer are mediated principally through well-defined regions in the broader lower domains, to form a structure that conforms well with that expected from the theory of quasi-equivalence with no significant conformational changes within the individual trimers. The VP3 layer determines the particle size and forms a rather smooth surface upon which the two-dimensional lattice of VP7 trimers is laid down.

Basak AK, Grimes JM, Gouet P, Roy P, Stuart DI. 1997. Structures of orbivirus VP7: implications for the role of this protein in the viral life cycle. Structure, 5 (7), pp. 871-883. | Show Abstract | Read more

BACKGROUND: Bluetongue virus (BTV) is the prototypical virus of the genus orbivirus in the family Reoviridae and causes an economically important disease in domesticated animals, such as sheep. BTV is larger and more complex than any virus for which comprehensive atomic level structural information is available. Its capsid is made primarily from four structural proteins two of which, VP3 and VP7, form a core which remains intact as the virus penetrates the host cell. Each core particle contains 780 copies of VP7. The architecture of the trimeric VP7 molecule has been revealed by crystallographic analysis and is unlike other viral coat proteins reported to date. RESULTS: Two new crystal structures of VP7 have been solved, one (a cleavage product) at close to atomic resolution and the other at lower resolution. The VP7 subunit consists of two domains. The smaller, 'upper', domain is exposed on the core surface and has the beta jelly-roll motif common to many capsid proteins. The second, 'lower', domain is composed of a bundle of alpha helices. The cleavage product comprises the upper domain, which forms a rigid invariant trimeric fragment. The lower resolution structure of the intact molecule indicates that the alpha-helical domain can rotate about the linker to the upper domain to adopt radically different orientations with respect to the threefold axis in the intact protein. CONCLUSIONS: The crystal structures of VP7 reveal a remarkable mix of rigidity and flexibility that may provide insights towards understanding how VP7 interacts with the other capsid proteins of different stoichiometries. These results suggest that substantial conformational changes in VP7 occur at some stage in the viral life cycle. Such changes may be related to the central role that VP7 is likely to play in cell attachment and membrane penetration.

Gao GF, Tormo J, Gerth UC, Wyer JR, McMichael AJ, Stuart DI, Bell JI, Jones EY, Jakobsen BK. 1997. Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2. Nature, 387 (6633), pp. 630-634. | Show Abstract | Read more

The dimeric cell-surface glycoprotein CD8 is crucial to the positive selection of cytotoxic T cells in the thymus. The homodimer CD8alpha(alpha) or the heterodimer alpha beta stabilizes the interaction of the T-cell antigen receptor (TCR) with major histocompatibility complex (MHC) class I/peptide by binding to the class I molecule. Here we report the crystal structure at 2.7 A resolution of a complex between CD8alpha(alpha) and the human MHC molecule HLA-A2, which is associated with peptide. CD8alpha(alpha) binds one HLA-A2/peptide molecule, interfacing with the alpha2 and alpha3 domains of HLA-A2 and also contacting beta2-microglobulin. A flexible loop of the alpha3 domain (residues 223-229) is clamped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody-antigen interaction, precluding the binding of a second MHC molecule. The position of the alpha3 domain is different from that in uncomplexed HLA-A2, being most similar to that in the TCR/Tax/HLA-A2 complex, but no conformational change extends to the MHC/peptide surface presented for TCR recognition. Although these shifts in alpha3 may provide a synergistic modulation of affinity, the binding of CD8 to MHC is clearly consistent with an avidity-based contribution from CD8 to TCR-peptide-MHC interactions.

Esnouf RM, Stuart DI, De Clercq E, Schwartz E, Balzarini J. 1997. Models which explain the inhibition of reverse transcriptase by HIV-1-specific (thio)carboxanilide derivatives. Biochem Biophys Res Commun, 234 (2), pp. 458-464. | Show Abstract | Read more

The (thio)carboxanilide derivatives are potent and selective inhibitors of HIV-1 reverse transcriptase (RT) and have a favourable antiviral activity spectrum. To understand better their mode of action, and to provide a structural basis for further improvement, models of RT complexed with four (thio)carboxanilide inhibitors (UC781, UC10, UC38 and UC84) have been constructed based on the X-ray structure of RT complexed with 9-chloro-TIBO. In the models, the protein conformation is similar to that of the RT-TIBO complex and the complexes are stabilised by hydrogen bonding between the inhibitors and the main chain oxygen of Lys101. Significant hydrophobic interactions include those with Leu100, Val106, Val179, Tyr188, Phe227, Leu234, and His235. The thiocarboxanilides UC781 and UC10 also make important hydrophobic interactions with Trp229. The models are consistent with the inhibitors' relative antiviral potencies and the observed resistance data. They further predict that mutations to Phe227, Trp229, or Leu234 might confer resistance. Since these are not observed, some constraining structural or functional role for these residues in the active enzyme is suggested.

Esnouf RM, Ren J, Hopkins AL, Ross CK, Jones EY, Stammers DK, Stuart DI. 1997. Unique features in the structure of the complex between HIV-1 reverse transcriptase and the bis(heteroaryl)piperazine (BHAP) U-90152 explain resistance mutations for this nonnucleoside inhibitor. Proc Natl Acad Sci U S A, 94 (8), pp. 3984-3989. | Show Abstract | Read more

The viral reverse transcriptase (RT) provides an attractive target in the search for anti-HIV therapies. The nonnucleoside inhibitors (NNIs) are a diverse set of compounds (usually HIV-1 specific) that function by distorting the polymerase active site upon binding in a nearby pocket. Despite being potent and of generally low toxicity, their clinical use has been limited by rapid selection for resistant viral populations. The 2.65-A resolution structure of the complex between HIV-1 RT and the bis(heteroaryl)piperazine (BHAP) NNI, 1-(5-methanesulfonamido-1H-indol-2-yl-carbonyl)-4- [3-(1-methyl-ethylamino) pyridinyl] piperazine (U-90152), reveals the inhibitor conformation and bound water molecules. The bulky U-90152 molecule occupies the same pocket as other NNIs, but the complex is stabilized quite differently, in particular by hydrogen bonding to the main chain of Lys-103 and extensive hydrophobic contacts with Pro-236. These interactions rationalize observed resistance mutations, notably Pro-236-Leu, which occurs characteristically for BHAPs. When bound, part of U-90152 protrudes into the solvent creating a channel between Pro-236 and the polypeptide segments 225-226 and 105-106, giving the first clear evidence of the entry mode for NNIs. The structure allows prediction of binding modes for related inhibitors [(altrylamino)piperidine-BHAPs] and suggests changes to U-90152, such as the addition of a 6 amino group to the pyridine ring, which may make binding more resilient to mutations in the RT. The observation of novel hydrogen bonding to the protein main chain may provide lessons for the improvement of quite different inhibitors.

Stuart DI, Jones EY. 1997. Cutting complexity down to size. Nature, 386 (6624), pp. 437-438. | Show Abstract | Read more

The proteasome is the most complex eukaryotic macromolecular assembly yet seen in fine detail. The structure reveals completely unexpected mechanisms by which the proteasome neatly chops up unwanted proteins for disposal or display to the immune system.

Hewat EA, Verdaguer N, Fita I, Blakemore W, Brookes S, King A, Newman J, Domingo E, Mateu MG, Stuart DI. 1997. Structure of the complex of an Fab fragment of a neutralizing antibody with foot-and-mouth disease virus: positioning of a highly mobile antigenic loop. EMBO J, 16 (7), pp. 1492-1500. | Show Abstract | Read more

Data from cryo-electron microscopy and X-ray crystallography have been combined to study the interactions of foot-and-mouth disease virus serotype C (FMDV-C) with a strongly neutralizing monoclonal antibody (mAb) SD6. The mAb SD6 binds to the long flexible GH-loop of viral protein 1 (VP1) which also binds to an integrin receptor. The structure of the virus-Fab complex was determined to 30 A resolution using cryo-electron microscopy and image analysis. The known structure of FMDV-C, and of the SD6 Fab co-crystallized with a synthetic peptide corresponding to the GH-loop of VP1, were fitted to the cryo-electron microscope density map. The SD6 Fab is seen to project almost radially from the viral surface in an orientation which is only compatible with monovalent binding of the mAb. Even taking into account the mAb hinge and elbow flexibility, it is not possible to model bivalent binding without severely distorting the Fabs. The bound GH-loop is essentially in what has previously been termed the 'up' position in the best fit Fab orientation. The SD6 Fab interacts almost exclusively with the GH-loop of VP1, making very few other contacts with the viral capsid. The position and orientation of the SD6 Fab bound to FMDV-C is in accord with previous immunogenic data.

Cited:

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Gao GF, Tormo J, Gerth UC, Wyer JR, McMichael AJ, Stuart DI, Bell JI, Jones EY, Jakobsen BK. 1997. Crystal structure of the complex between CD8αα human and HLA-A2 Nature, 387 (6633), pp. 630-634. | Show Abstract | Read more

The dimeric cell-surface glycoprotein CD8 is crucial to the positive selection of cytotoxic T cells in the thymus. The homodimer CD8αα or the heterodimer αβ stabilizes the interaction of the T-cell antigen receptor (TCR) with major histocompatibility complex (MHC) class l/peptide by binding to the class I molecule. Here we report the crystal structure at 2.7 Å resolution of a complex between CD8αα and the human MHC molecule HLA-A2, which is associated with peptide. CD8αα binds one HLA-A2/peptide molecule, interfacing with the α2 and α3 domains of HLA-A2 and also contacting β2- microglobulin. A flexible loop of the α3 domain (residues 223-229) is damped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody-antigen interaction, precluding the binding of a second MHC molecule. The position of the α3 domain is different from that in uncomplexed HLA-A2 (refs 3, 4), being most similar to that in the TCR/Tax/HLA-A2 complex, but no conformational change extends to the MHC/peptide surface presented for TCR recognition. Although these shifts in α3 may provide a synergistic modulation of affinity, the binding of CD8 to MHC is dearly consistent with an avidity-based contribution from CD8 to TCR-peptide-MHC interactions.

Reid SW, McAdam S, Smith KJ, Klenerman P, O'Callaghan CA, Harlos K, Jakobsen BK, McMichael AJ, Bell JI, Stuart DI, Jones EY. 1996. Antagonist HIV-1 Gag peptides induce structural changes in HLA B8. J Exp Med, 184 (6), pp. 2279-2286. | Show Abstract | Read more

In the cellular immune response, recognition by CTL-TCRs of viral antigens presented as peptides by HLA class I molecules, triggers destruction of the virally infected cell (Townsend, A.R.M., J. Rothbard, F.M. Gotch, G. Bahadur, D. Wraith, and A.J. McMichael. 1986. Cell. 44:959-968). Altered peptide ligands (APLs) which antagonise CTL recognition of infected cells have been reported (Jameson, S.C., F.R. Carbone, and M.J. Bevan. 1993. J. Exp. Med. 177:1541-1550). In one example, lysis of antigen presenting cells by CTLs in response to recognition of an HLA B8-restricted HIV-1 P17 (aa 24-31) epitope can be inhibited by naturally occurring variants of this peptide, which act as TCR antagonists (Klenerman, P., S. Rowland Jones, S. McAdam, J. Edwards, S. Daenke, D. Lalloo, B. Koppe, W. Rosenberg, D. Boyd, A. Edwards, P. Giangrande, R.E. Phillips, and A. McMichael. 1994. Nature (Lond.). 369:403-407). We have characterised two CTL clones and a CTL line whose interactions with these variants of P17 (aa 24-31) exhibit a variety of responses. We have examined the high resolution crystal structures of four of these APLs in complex with HLA B8 to determine alterations in the shape, chemistry, and local flexibility of the TCR binding surface. The variant peptides cause changes in the recognition surface by three mechanisms: changes contributed directly by the peptide, effects transmitted to the exposed peptide surface, and induced effects on the exposed framework of the peptide binding groove. While the first two mechanisms frequently lead to antagonism, the third has more profound effects on TCR recognition.

Jackson T, Ellard FM, Ghazaleh RA, Brookes SM, Blakemore WE, Corteyn AH, Stuart DI, Newman JW, King AM. 1996. Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate. J Virol, 70 (8), pp. 5282-5287. | Show Abstract

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.

Robinson RC, Radziejewski C, Stuart DI, Jones EY. 1996. Crystals of the neurotrophins. Protein Sci, 5 (5), pp. 973-977. | Show Abstract | Read more

The neurotrophins show a high degree of amino acid sequence homology, share similar solution properties, and display distinct but parallel functionalities. Here we report the crystallization and preliminary X-ray characterization of three neurotrophins: brain-derived neurotrophin, neurotrophin 3, and the heterodimer between brain-derived neurotrophin and neurotrophin 4. These findings are related to other published crystal parameters for neurotrophins, leading to the observation that, although crystal packing is highly variant, neurotrophins share common solubilities with respect to crystal growth.

Hopkins AL, Ren J, Esnouf RM, Willcox BE, Jones EY, Ross C, Miyasaka T, Walker RT, Tanaka H, Stammers DK, Stuart DI. 1996. Complexes of HIV-1 reverse transcriptase with inhibitors of the HEPT series reveal conformational changes relevant to the design of potent non-nucleoside inhibitors. J Med Chem, 39 (8), pp. 1589-1600. | Show Abstract | Read more

Crystal structures of HIV-1 reverse transcriptase (RT) complexed with a range of chemically diverse non-nucleoside inhibitors (NNIs) have shown a single pocket in which the inhibitors bind and details of the inhibitor-protein interactions. To delineate the structural requirements for an effective inhibitor, we have determined the structures of three closely related NNIs which vary widely in their potencies. Crystal structures of HIV-1 RT complexed with two very potent inhibitors, MKC-442 and TNK-651, at 2.55 angstroms resolution complement our previous analysis of the complex with the less effective inhibitor, HEPT. These structures reveal conformational changes which correlate with changes in potency. We suggest that a major determinant of increased potency in the analogues of HEPT is an improved interaction between residue Tyr181 in the protein and the 6-benzyl ring of the inhibitors which stabilizes the structure of the complex. This arises through a conformational switching of the protein structure triggered by the steric bulk of the 5-substituent of the inhibitor pyrimidine ring.

Reid SW, Smith KJ, Jakobsen BK, O'Callaghan CA, Reyburn H, Harlos K, Stuart DI, McMichael AJ, Bell JI, Jones EY. 1996. Production and crystallization of MHC class I B allele single peptide complexes. FEBS Lett, 383 (1-2), pp. 119-123. | Show Abstract | Read more

Major histocompatibility complex class I B alleles, HLA B8, B53 and B3501 have been cloned, expressed, refolded and crystallized in specific complexes with a number of different 8-mer and 9-mer peptides. For some of these crystallization was initiated by cross-seeding between different B allele complexes. All crystallize in the space group P212121, with similar unit cell dimensions of approximately 52 A X 81 A X 112 A, contain one complex per asymmetric unit and diffract to approximately 2.0 A resolution.

Smith KJ, Reid SW, Stuart DI, McMichael AJ, Jones EY, Bell JI. 1996. An altered position of the alpha 2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501. Immunity, 4 (3), pp. 203-213. | Show Abstract | Read more

The crystal structure of the human major histocompatibility complex class I B allele HLA B*3501 complexed with the 8-mer peptide epitope HIV1 Nef 75-82 (VPLRPMTY) has been determined at 2.0 angstrom resolution. Comparison with the crystal structure of the closely related allele HLA B*5301 reveals the structural basis for the tyrosine specificity of the B*3501 F pocket. The structure also reveals a novel conformation of the 8-mer peptide within the binding groove. The positions of the peptide N and C termini are nonstandard, but the classic pattern of hydrogen bonding to nonpolymorphic MHC class I residues is maintained, at the N terminus by addition of a water molecule, and at the C terminus by a substantial shift in the alpha 2 helix.

Smith KJ, Reid SW, Harlos K, McMichael AJ, Stuart DI, Bell JI, Jones EY. 1996. Bound water structure and polymorphic amino acids act together to allow the binding of different peptides to MHC class I HLA-B53. Immunity, 4 (3), pp. 215-228. | Show Abstract | Read more

The structure of the human MHC class I molecule HLA-B53 complexed to two nonameric peptide epitopes (from the malaria parasite P. falciparum and the HIV2 gag protein) has been determined by X-ray crystallography at 2.3 angstrom resolution. The structures reveal the architecture of a Pro-specific B pocket common to many HLA-B alleles. Relative to other alleles, the B53 peptide-binding groove is widened by a significant (up to 1.25 angstrom) shift in the position of the alpha 1 helix. Within this groove, bound water molecules, acting in concert with the side chains of polymorphic residues, provide the functional malleability of the MHC, which enables the high affinity/low specificity binding of multiple peptide epitopes.

Rao Z, Belyaev AS, Fry E, Roy P, Jones IM, Stuart DI. 1995. Crystal structure of SIV matrix antigen and implications for virus assembly. Nature, 378 (6558), pp. 743-747. | Show Abstract | Read more

Simian immunodeficiency virus (SIV) is closely related to human immunodeficiency virus (HIV), their matrix antigens (MAs) sharing some 50% sequence identity. MA is a component of Pr55Gag, the sole protein required for assembly of the virion shell. MA targets Pr55 to the plasma membrane, and facilitates incorporation of the virus envelope protein and assembly of the Pr55Gag shell. Cleavage of Pr55 by the viral protease produces the mature protein of relative molecular mass 17-18K, which underlies the host-derived membrane and is important in both virus entry and nuclear localization of the virion core. Here we report the crystal structure of SIV MA. The molecule forms a trimer consistent with oligomerization in vitro, the observed virion architecture, and various biological properties of MA.

Stuart DI, Jones EY. 1995. Recognition at the cell surface: recent structural insights. Curr Opin Struct Biol, 5 (6), pp. 735-743. | Show Abstract | Read more

In the past few years, structural biology has begun to reveal details of some of the receptors and associated interactions responsible for protein-mediated recognition events at the cell surface. Recent data span the fields of cytokine-receptor interactions, cell adhesion molecules and viral infection.

Ren J, Esnouf R, Hopkins A, Ross C, Jones Y, Stammers D, Stuart D. 1995. The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for inhibitor design. Structure, 3 (9), pp. 915-926. | Show Abstract | Read more

BACKGROUND: HIV reverse transcriptase (RT) is a key target of anti-AIDS therapies. Structural studies of HIV-1 RT, unliganded and complexed with different non-nucleoside inhibitors (NNIs), have pointed to a common mode of binding and inactivation through distortion of the polymerase catalytic site by NNIs containing two hinged rings. The mode of binding of the TIBO family of inhibitors is of interest because these compounds do not fit the two-hinged-ring model. RESULTS: The structure of HIV-1 RT complexed with 9-chloro-TIBO (R82913) has been determined at 2.6 A resolution. As reported for the lower resolution analysis of another TIBO compound, this inhibitor binds at the same site as other NNIs, but our higher resolution study reveals the Cl-TIBO is distorted from the conformation seen in crystals of the inhibitor alone. This allows Cl-TIBO to mimic the binding of NNIs containing two hinged rings. Inhibitor-protein interactions are again predominantly hydrophobic and the protein conformation corresponds to that seen in complexes with other tight-binding NNIs. CONCLUSIONS: Although Cl-TIBO is chemically very different from other NNIs, it achieves remarkable spatial equivalence and shape complementarity with other NNIs on binding to RT. Comparison of the different RT-NNI complexes suggests modifications to the TIBO group of inhibitors which might enhance their binding and hence, potentially, their therapeutic efficacy.

Robinson RC, Grey LM, Staunton D, Stuart DI, Heath JK, Jones EY. 1995. The crystal structure of murine leukemia inhibitory factor. Ann N Y Acad Sci, 762 pp. 179-187. | Show Abstract

We have determined the structure of murine leukemia inhibitory factor (LIF) by X-ray crystallography at 2.0 A resolution. The current crystal structure comprises native LIF residues 9 to 180 with 40 ordered water molecules. For this model the R value (with a bulk solvent correction) is 18.6% on all data from 20.0 A to 2.0 A with stereochemistry typified by root mean square deviations from ideal bond lengths of 0.015 A. The mainchain fold conforms to the four alpha-helix bundle topology previously observed for several members of the hematopoietic cytokine family. Of these, LIF shows closest structural homology to granulocyte colony stimulating factor and growth hormone. Sequence alignments for the functionally related molecules oncostatin M and ciliary neurotrophic factor, when mapped to the LIF structure, indicate regions of conserved structural and surface character. Analysis of published mutagenesis data implicate two regions of receptor interaction which are located in the fourth helix and the preceding loop. A model for receptor binding based on the structure of the growth hormone ligand/receptor complex requires additional, novel features to account for these data.

Spraggon G, Phillips C, Nowak UK, Ponting CP, Saunders D, Dobson CM, Stuart DI, Jones EY. 1995. The crystal structure of the catalytic domain of human urokinase-type plasminogen activator. Structure, 3 (7), pp. 681-691. | Show Abstract | Read more

BACKGROUND: Urokinase-type plasminogen activator (u-PA) promotes fibrinolysis by catalyzing the conversion of plasminogen to the active protease plasmin via the cleavage of a peptide bond. When localized to the external cell surface it contributes to tissue remodelling and cellular migration; inhibition of its activity impedes the spread of cancer. u-PA has three domains: an N-terminal receptor-binding growth factor domain, a central kringle domain and a C-terminal catalytic protease domain. The biological roles of the fibrinolytic enzymes render them therapeutic targets, however, until now no structure of the protease domain has been available. Solution of the structure of the u-PA serine protease was undertaken to provide such data. RESULTS: The crystal structure of the catalytic domain of recombinant, non-glycosylated human u-PA, complexed with the inhibitor Glu-Gly-Arg chloromethyl ketone (EGRcmk), has been determined at a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 22.4% on all data (20.4% on data > 3 sigma). The enzyme has the expected topology of a trypsin-like serine protease. CONCLUSIONS: The enzyme has an S1 specificity pocket similar to that of trypsin, a restricted, less accessible, hydrophobic S2 pocket and a solvent-accessible S3 pocket which is capable of accommodating a wide range of residues. The EGRcmk inhibitor binds covalently at the active site to form a tetrahedral hemiketal structure. Although the overall structure is similar to that of homologous serine proteases, at six positions insertions of extra residues in loop regions create unique surface areas. One of these loop regions is highly mobile despite being anchored by the disulphide bridge which is characteristic of a small subset of serine proteases namely tissuetype plasminogen activator, Factor XII and Complement Factor I.

Burroughs JN, Grimes JM, Mertens PP, Stuart DI. 1995. Crystallization and preliminary X-ray analysis of the core particle of bluetongue virus. Virology, 210 (1), pp. 217-220. | Show Abstract | Read more

Core particles of bluetongue virus serotype 1 (South Africa) have been crystallized. The crystals, which grow up to 0.8 mm in diameter, belong to a primitive orthorhombic space group and have point group symmetry 222. The unit cell dimensions are 754 x 796 x 823 A3 and the crystallographic asymmetric unit contains one-half of a core particle. The best crystals diffract strongly to 4.8 A Bragg spacings, which is the maximum resolution to which we can measure data with the detectors available, suggesting that useful diffraction extends well beyond this. Core particles of serotype 10 have also been crystallized but the crystals have yet to be analyzed by X-ray diffraction.

Robinson RC, Radziejewski C, Stuart DI, Jones EY. 1995. Structure of the brain-derived neurotrophic factor/neurotrophin 3 heterodimer. Biochemistry, 34 (13), pp. 4139-4146. | Show Abstract | Read more

The development and sustenance of specific neuronal populations in the peripheral and central nervous systems are controlled through the binding of neurotrophic factors to high-affinity cell surface receptors. The neurotrophins (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT3; and neurotrophin 4, NT4) are dimeric molecules which share approximately 50% sequence identity. The crystal structure of the murine NGF homodimer [McDonald et al. (1991) Nature 354, 411-414] indicated that the dimer interface corresponds to regions of high sequence conservation throughout the neurotrophin family. This potential compatibility was duly exploited for the production in vitro of noncovalent heterodimers between the different neurotrophins [Radziejewski, C., & Robinson, R.C. (1993) Biochemistry 32, 13350-13356; Jungbluth et al. (1994) Eur. J. Biochem. 221, 677-685]. Here, we report the X-ray structure at 2.3 A resolution of one such heterodimer, between human BDNF, and human NT3. The NGF, BDNF, and NT3 protomers share the same topology and are structurally equivalent in regions which contribute to the dimer interface in line with the propensity of the neurotrophins to form heterodimers. Analysis of the structure of regions of the BDNF/NT3 heterodimer involved in receptor specificity led us to conclude that heterodimer binding to p75 involves distant binding sites separately located on each protomer of the heterodimer. In contrast, heterodimer interactions with the trk receptors probably utilize hybrid binding sites comprised of residues contributed by both protomers in the heterodimer. The existence of such hybrid binding sites for the trk receptor provides an explanation for the lower activity of the BDNF/NT3 heterodimer in comparison to the homodimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Esnouf R, Ren J, Ross C, Jones Y, Stammers D, Stuart D. 1995. Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors. Nat Struct Biol, 2 (4), pp. 303-308. | Show Abstract | Read more

The structure of unliganded HIV-1 reverse transcriptase has been determined at 2.35 A resolution and refined to an R-factor of 0.219 (for all data) with good stereochemistry. The unliganded structure was produced by soaking out a weak binding non-nucleoside inhibitor, HEPT, from pregrown crystals. Comparison with the structures of four different RT and non-nucleoside inhibitor complexes reveals that only minor domain rearrangements occur, but there is a significant repositioning of a three-stranded beta-sheet in the p66 subunit (containing the catalytic aspartic acid residues 110, 185 and 186) with respect to the rest of the polymerase site. This suggests that NNIs inhibit RT by locking the polymerase active site in an inactive conformation, reminiscent of the conformation observed in the inactive p51 subunit.

Ren J, Esnouf R, Garman E, Somers D, Ross C, Kirby I, Keeling J, Darby G, Jones Y, Stuart D. 1995. High resolution structures of HIV-1 RT from four RT-inhibitor complexes. Nat Struct Biol, 2 (4), pp. 293-302. | Show Abstract | Read more

We have determined the structures of four complexes of HIV-1 reverse transcriptase with non-nucleoside inhibitors, three fully refined at high resolution. The highest resolution structure is of the RT-nevirapine complex which has an R-factor of 0.186 and a root-mean-square bond length deviation of 0.015 A for all data to 2.2 A. The structures reveal a common mode of binding for these chemically diverse compounds. The common features of binding are largely hydrophobic interactions and arise from induced shape complementarity achieved by conformational rearrangement of the enzyme and conformational/configurational rearrangement of the compounds.

Jones EY, Harlos K, Bottomley MJ, Robinson RC, Driscoll PC, Edwards RM, Clements JM, Dudgeon TJ, Stuart DI. 1995. Crystal structure of an integrin-binding fragment of vascular cell adhesion molecule-1 at 1.8 A resolution. Nature, 373 (6514), pp. 539-544. | Show Abstract | Read more

The cell-surface glycoprotein vascular cell adhesion molecule-1 (VCAM-1; ref. 1) mediates intercellular adhesion by specific binding to the integrin very-late antigen-4 (VLA-4, alpha 4 beta 1; ref. 3). VCAM-1, with the intercellular adhesion molecules ICAM-1, ICAM-2, ICAM-3 and the mucosal vascular addressin MAd-CAM-1, forms an integrin-binding subgroup of the immunoglobulin superfamily. In addition to their clinical relevance in inflammation, these molecules act as cellular receptors for viral and parasitic agents. The predominant form of VCAM-1 in vivo has an amino-terminal extracellular region comprising seven immunoglobulin-like domains. Functional studies have identified a conserved integrin-binding motif in domains 1 and 4, variants of which are present in the N-terminal domain of all members of the immunoglobulin superfamily subgroup. We report here the crystal structure of a VLA-4-binding fragment composed of the first two domains of VCAM-1. The integrin-binding motif (Q38IDSPL) is highly exposed and forms the N-terminal region of the loop between beta-strands C and D of domain 1. This motif exhibits a distinctive conformation which we predict will be common to all the integrin-binding IgSF molecules. These, and additional data, map VLA-4 binding to the face of the CFG beta-sheet, the surface previously identified as the site for intercellular adhesive interactions between members of the immunoglobulin superfamily.

Davis SJ, Davies EA, Barclay AN, Daenke S, Bodian DL, Jones EY, Stuart DI, Butters TD, Dwek RA, van der Merwe PA. 1995. Ligand binding by the immunoglobulin superfamily recognition molecule CD2 is glycosylation-independent. J Biol Chem, 270 (1), pp. 369-375. | Show Abstract | Read more

The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-A resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.

Curry S, Abrams CC, Fry E, Crowther JC, Belsham GJ, Stuart DI, King AM. 1995. Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids. J Virol, 69 (1), pp. 430-438. | Show Abstract

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.

Basak AK, Stuart DI, Nikura T, Bishop DH, Kelly DC, Fearn A, Titball RW. 1994. Purification, crystallization and preliminary X-ray diffraction studies of alpha-toxin of Clostridium perfringens. J Mol Biol, 244 (5), pp. 648-650. | Show Abstract | Read more

Alpha-toxin of Clostridium perfringens, cloned in Escherichia coli, has been purified and crystallized from ammonium sulphate using the hanging drop vapour diffusion method at 20 degrees C. The crystals diffract to a minimum Bragg spacing of 2.7 A, belong to the space group R32 (with a = b = 153.3 A, c = 95.4 A, alpha = beta = 90 degrees and gamma = 120 degrees) and contain a single polypeptide chain in the crystallographic unit.

Bottomley MJ, Robinson RC, Driscoll PC, Harlos K, Stuart DI, Aplin RT, Clements JM, Jones EY, Dudgeon TJ. 1994. Crystallization and preliminary X-ray diffraction characterisation of both a native and selenomethionyl VLA-4 binding fragment of VCAM-1. J Mol Biol, 244 (4), pp. 464-468. | Show Abstract | Read more

Soluble fragments of the extracellular region of vascular cell adhesion molecule 1 (VCAM-1) expressed in Escherichia coli retain functional adhesive activity. An integrin (VLA-4) binding fragment consisting of the N-terminal two immunoglobulin-like domains (VCAM-d1,2) has been crystallized. The crystals belong to space group P2(1)2(1)2(1) with cell dimensions of a = 52.7 A, b = 66.5 A, c = 113.2 A and contain two molecules in the crystallographic asymmetric unit. A batch of protein produced in the standard E. coli strain (HW1110), but grown in the presence of selenomethionine enriched media, showed 85% incorporation of selenium in place of sulphur at methionine residues. The selenomethionyl VCAM-d1,2 was crystallized by microseeding techniques initially using the native crystals for nucleation. Both native and selenomethionyl crystals diffract X-rays to a minimum Bragg spacing of 1.8 A.

Stammers DK, Somers DO, Ross CK, Kirby I, Ray PH, Wilson JE, Norman M, Ren JS, Esnouf RM, Garman EF. 1994. Crystals of HIV-1 reverse transcriptase diffracting to 2.2 A resolution. J Mol Biol, 242 (4), pp. 586-588. | Show Abstract | Read more

Reverse transcriptase (RT) from the human immunodeficiency virus type 1 has been crystallized in four closely related forms, the best of which diffract X-rays to 2.2 A resolution. The RT was crystallized as a complex with a non-nucleoside inhibitor, either nevirapine or a nevirapine analogue. Crystals grew from 6% PEG 3400 buffered at pH 5. These were of space group P2(1)2(1)2(1) with unit cell parameters a = 147 A, b = 112 A, c = 79 A (form A), with one RT heterodimer in the asymmetric unit. Changes in unit cell parameters and degree of crystalline order were observed on soaking pregrown crystals in various solutions, giving three further sets of unit cells. These were a = 143 A, b = 112, A, c = 79 A (form B), a = 141 A, b = 111 A, c = 73 A (form C), a = 143 A, b = 117 A, c = 66.5 A (form D). The last two forms diffract X-rays to 2.2 A resolution. Structure determinations of these latter crystal forms of RT should give a detailed atomic model for this therapeutically important drug target.

Harlos K, Martin DM, O'Brien DP, Jones EY, Stuart DI, Polikarpov I, Miller A, Tuddenham EG, Boys CW. 1994. Crystal structure of the extracellular region of human tissue factor. Nature, 370 (6491), pp. 662-666. | Show Abstract | Read more

Tissue factor is a cell-surface glycoprotein receptor which initiates the blood coagulation cascade after vessel injury by interacting with blood clotting factor VII/VIIa and which is implicated in various pathological processes. When bound to tissue factor, factor VII is readily converted to the active protease factor VIIa by trace amounts of factors Xa, IXa or VIIa. Human tissue factor consists of 263 residues, the first 219 of which comprise the extracellular region. We have determined the crystal structure of the extracellular region at a resolution of 2.2 A. Tissue factor consists of two immunoglobulin-like domains associated through an extensive, novel, interdomain interface region. The binding site for factor VII lies at the interface region and involves residues from domain 1 and an extended loop (binding 'finger') of domain 2. This is the first reported structure of a representative of the class 2 cytokine receptor family, which also includes interferon-alpha, interferon-gamma (refs 2, 3) and interleukin-10 (ref. 4) receptors.

Bodian DL, Jones EY, Harlos K, Stuart DI, Davis SJ. 1994. Crystal structure of the extracellular region of the human cell adhesion molecule CD2 at 2.5 A resolution. Structure, 2 (8), pp. 755-766. | Show Abstract | Read more

BACKGROUND: The T-lymphocyte antigen CD2 is an adhesion molecule implicated in immune responses in vivo. The extracellular regions of the human and rat homologues of CD2 share only 45% sequence identity and bind different protein ligands. Comparison of the human and rat soluble CD2 (sCD2) structures should provide insights into the structural basis of cell surface recognition. RESULTS: We therefore determined the crystal structure of a form of human sCD2 with single N-acetylglucosamine residues at each glycosylation site to 2.5 A resolution with an R-factor of 19.3%. It is composed of two immunoglobulin superfamily domains similar to those of rat sCD2, but the relative orientation of the domains in the two homologues differs by up to 20 degrees. An interaction involving the flat, highly charged, ligand binding GFCC'C" faces of crystallographically related human sCD2 molecules duplicates, in a different lattice, that observed in the rat sCD2 crystals. CONCLUSIONS: Intramolecular flexibility appears to be a conserved feature of CD2. The head-to-head interaction between molecules represents a general model for interactions between adhesion molecules of this structural class. Ligand specificity may be influenced by the distribution of charged residues on the binding face.

Acharya KR, Stuart DI, Phillips DC, McKenzie HA, Teahan CG. 1994. Models of the three-dimensional structures of echidna, horse, and pigeon lysozymes: calcium-binding lysozymes and their relationship with alpha-lactalbumins. J Protein Chem, 13 (6), pp. 569-584. | Show Abstract | Read more

Similarities in amino acid sequences, three-dimensional structures, and the exon-intron patterns of their genes have indicated that c-type lysozymes and alpha-lactalbumins are homologous proteins, i.e., descended by divergent evolution from a common ancestor. Like the alpha-lactalbumins, echidna milk, horse milk, and pigeon eggwhite lysozymes all bind Ca(II). Models of their three-dimensional structures, based on their amino acid sequences and the known crystal structures of domestic hen eggwhite and human lysozymes and baboon and human alpha-lactalbumins, have been built. The several structures have been compared and their relationships discussed.

Robinson RC, Grey LM, Staunton D, Vankelecom H, Vernallis AB, Moreau JF, Stuart DI, Heath JK, Jones EY. 1994. The crystal structure and biological function of leukemia inhibitory factor: implications for receptor binding. Cell, 77 (7), pp. 1101-1116. | Show Abstract | Read more

The structure of murine leukemia inhibitory factor (LIF) has been determined by X-ray crystallography at 2.0 A resolution. The main chain fold conforms to the four alpha-helix bundle topology previously observed for several members of the hematopoietic cytokine family. Of these, LIF shows closest structural homology to granulocyte colony-stimulating factor and growth hormone (GH). Sequence alignments for the functionally related molecules oncostatin M and ciliary neurotrophic factor, when mapped to the LIF structure, indicate regions of conserved surface character. Analysis of the biological function and receptor specificity of a series of human-mouse LIF chimeras implicate two regions of receptor interaction that are located in the fourth helix and the preceding loop. A model for receptor binding based on the structure of the GH ligand-receptor complex requires additional, novel features to account for these data.

Lea S, Hernández J, Blakemore W, Brocchi E, Curry S, Domingo E, Fry E, Abu-Ghazaleh R, King A, Newman J. 1994. The structure and antigenicity of a type C foot-and-mouth disease virus. Structure, 2 (2), pp. 123-139. | Show Abstract | Read more

BACKGROUND: Picornaviruses are responsible for a wide range of mammalian diseases and, in common with other RNA viruses, show considerable antigenic variation. Foot-and-mouth disease viruses (FMDVs) constitute one genus of the picornavirus family and are classified into seven serotypes, each of which shows considerable intratypic variation. This antigenic variation leads to continuing difficulties in controlling the disease. To date the structure of only one serotype, O, has been reported. RESULTS: The three-dimensional structure of a serotype C (isolate C-S8c1) FMDV, has been determined crystallographically at 3.5 A resolution. The main chain conformation of the virion is very similar to that of type O1 virus. The immunodominant G-H loop of VP1, the presumed site of cell attachment, is disordered in both types of virus indicating a functional role for flexibility of this region. There are significant changes in the structure of other antigenic loops and in some internal regions involved in protomer-protomer contacts, including the entire amino-terminal portion of VP2, described here for the first time for a picornavirus. Antigenic sites have been identified by genetic and peptide mapping methods, and located on the capsid. The data reveal a major new discontinuous antigenic site (site D) which is located near to the three-fold axis and involves residues of VP1, VP2 and VP3 which lie adjacent to each other on the capsid. CONCLUSION: In FMDV type C, amino acid substitutions seen in mutants that are resistant to neutralization by monoclonal antibodies (MAbs) map to predominantly surface-oriented residues with solvent-accessible side-chains not involved in interactions with other amino acids, whereas residues which are accessible but not substituted are found to be more frequently involved in protein-protein interactions. This provides a molecular interpretation for the repeated isolation of the same amino acid substitutions in MAb-resistant variants, an observation frequently made with RNA viruses. This first comparison of two FMDV serotypes shows how subtle changes at antigenic sites are sufficient to cause large changes in antigenic specificity between serotypes.

REES D, GRANT R, RAO Z, STUART D. 1994. THE N-TERMINAL DOMAIN OF TALIN - INITIAL STRUCTURAL AND FUNCTIONAL-STUDIES JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 262-262.

Morgunova EYu, Dauter Z, Fry E, Stuart DI, Stel'mashchuk VYa, Mikhailov AM, Wilson KS, Vainshtein BK. 1994. The atomic structure of Carnation Mottle Virus capsid protein. FEBS Lett, 338 (3), pp. 267-271. | Show Abstract | Read more

The structure of the Carnation Mottle Virus (CMtV) capsid protein has been determined at 3.2 A resolution by the method of molecular replacement. Three-dimensional data were collected from a small number of crystals (sp.g. I23, a = 382.6 A) using the synchrotron radiation with an image plate as detector. The coordinates of Tomato Bushy Stunt Virus (TBSV) were used as a searching model. Refinement of the coordinates of 7,479 non-hydrogen atoms performed by the program XPLOR, has led to an R-factor of 18.3%. It was found that the amino acid chain fold of capsid protein is very similar to that in other icosahedral viruses. However, there are some differences in the contact regions between protein subunits and also the lack of the beta-annulus around the 3-fold icosahedral axes. The structural and biochemical results lead us to consider an alternative assembly pathway.

Tavernor AS, Kydd JH, Bodian DL, Jones EY, Stuart DI, Davis SJ, Butcher GW. 1994. Expression cloning of an equine T-lymphocyte glycoprotein CD2 cDNA. Structure-based analysis of conserved sequence elements. Eur J Biochem, 219 (3), pp. 969-976. | Show Abstract | Read more

An equine CD2 cDNA has been isolated by monoclonal antibody screening of a T-lymphocyte cDNA library. The cDNA contained an open reading frame of 1041 bp encoding a translated product of 347 amino acids. Northern blotting analysis revealed a single mRNA species expressed in spleen, thymus and activated peripheral lymphocytes. The predicted amino acid sequence has 50-65% identity with the human, rat and mouse CD2 sequences with greatest similarity shared with the human homologue. Evolutionarily conserved structural and functional domains in CD2 were identified by comparing the sequences of the equine, human, mouse and rat CD2 homologues in the context of the recently derived crystal structure of rat soluble CD2 [Jones, E. Y., Davis, S. J., Williams, A. F., Harlos, K. & Stuart, D. I. (1992) Nature 360, 232-239]. The key conserved features of the extracellular region included core residues necessary to preserve the structural integrity of the molecule, residues in the linker region likely to maintain the unique domain organization of CD2, an array of highly charged residues in the putative ligand-binding face of the molecule and glycosylation-signal distributions that render the putative ligand-binding GFCC'C" face of domain 1 relatively unhindered by glycosylation.

Ren J, Wang Y, Dong Y, Stuart DI. 1994. The N-glycosidase mechanism of ribosome-inactivating proteins implied by crystal structures of alpha-momorcharin. Structure, 2 (1), pp. 7-16. | Show Abstract | Read more

BACKGROUND: alpha-Momorcharin (alpha MMC) is a type I ribosome-inactivating protein. It inhibits protein synthesis by hydrolytically removing a specific adenine residue from a highly conserved, single-stranded loop of rRNA. RESULTS: Here we describe the determination and refinement of the crystal structures of alpha MMC in the native state and in complexes with the product, adenine, and a substrate analogue, formycin 5'-monophosphate (FMP) at high resolution. Both adenine and the base of FMP are tightly bound; the ribose of bound FMP adopts a strained, high-energy conformation, which may mimic the structure of the transition state. CONCLUSIONS: These structures indicate that residues Tyr70, Glu160 and Arg163 of alpha MMC are the most critical for catalysis. We propose that the strained conformation of the ribose in the target adenosine weakens the glycoside bond. Partial protonation mediated by Arg163 then facilitates N-glycoside bond cleavage, leading to the formation of an oxycarbonium ion intermediate which is stabilized by the negatively-charged Glu160. Tyr70 adopts subtly different conformations in the three structures implying that it may be important in substrate recognition and perhaps catalysis.

Acharya KR, Passalacqua EF, Jones EY, Harlos K, Stuart DI, Brehm RD, Tranter HS. 1994. Structural basis of superantigen action inferred from crystal structure of toxic-shock syndrome toxin-1. Nature, 367 (6458), pp. 94-97. | Show Abstract | Read more

Superantigens stimulate T cells bearing particular T-cell receptor V beta sequences, so they are extremely potent polyclonal T-cell mitogens. T-cell activation is preceded by binding of superantigens to class II major histocompatibility complex (MHC) molecules. To further the structural characterization of these interactions, the crystal structure of a toxin associated with toxic-shock syndrome, TSST-1, which is a microbial superantigen, has been determined at 2.5 A resolution. The N- and C-terminal domains of the structure both contain regions involved in MHC class II association; the C-terminal domain is also implicated in binding the T-cell receptor. Despite low sequence conservation, the TSST-1 topology is similar to the structure reported for the superantigen staphylococcal enterotoxin B4. But TSST-1 lacks several of the structural features highlighted as central to superantigen activity in the staphylococcal enterotoxin B and we therefore reappraise the structural basis of superantigen action.

Basak AK, Baker DP, Fewson CA, Stuart DI. 1993. Preliminary crystallographic study of D(-)-mandelate dehydrogenase from Rhodotorula graminis. J Mol Biol, 233 (4), pp. 781-783. | Show Abstract | Read more

NAD+ dependent D(-)-mandelate dehydrogenase from the yeast Rhodotorula graminis strain KGX 39 has been crystallized in three different forms using the hanging drop vapour diffusion method at 15 to 20 degrees C. Type I crystals belong to space group P222(1), P22(1)2(1) or P2(1)2(1)2(1) with a = 100.3 A, b = 117.4 A, c = 80.4 A and are likely to contain a dimer in the crystallographic asymmetric unit. They diffract to dmin = 3.0 A. Type II crystals belong to space group P22(1)2(1) or P2(1)2(1)2(1) with a = 187.8 A, b = 122.9 A, c = 72.1 A and contain probably two dimers in the crystallographic asymmetric unit. They diffract to dmin = 1.8 A. Type III crystals belong to space group P2(1)2(1)2(1) with a = 109.6, b = 52.0 A, c = 145.7 A, and are likely to contain a dimer in the crystallographic asymmetric unit. They diffract at least to dmin = 2.5 A.

STUART D, JONES E. 1993. WEISSENBERG DATA-COLLECTION FOR MACROMOLECULAR CRYSTALLOGRAPHY CURRENT OPINION IN STRUCTURAL BIOLOGY, 3 (5), pp. 737-740. | Show Abstract | Read more

The Weissenberg method has recently emerged as a viable technique in protein crystallography with the demonstration of fast, efficient X-ray diffraction data collection from a variety of protein crystals by this method at the Photon Factory (Tskuba, Japan). This review assesses the technique in the context of current results and future developments. © 1993.

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Ren J, Stuart DI, Acharya KR. 1993. α-lactalbumin possesses a distinct zinc binding site Journal of Biological Chemistry, 268 (26), pp. 19292-19298. | Show Abstract

It has been proposed that the binding of Zn2+ to α-lactalbumin switches the conformation to one akin to a state intermediate in the folding of the protein. However, the high resolution x-ray crystal structure of human α-lactalbumin-Zn2+ complex at 1.7-Å resolution (pH 7.6) does not reveal any significant change in conformation from the native state. The Zn2+ ion binds specifically in the "cleft" of α-lactalbumin (the region which forms the active site of the homologous protein lysozyme). This may suggest a possible role for Zn2+ binding in lactose synthase complex. The coordination of the Zn2+ ion involves a symmetry-related molecule in the crystal, the crystal contacts being stabilized by a SO42- ion bound at the interface between three molecules.

Ren J, Stuart DI, Acharya KR. 1993. Alpha-lactalbumin possesses a distinct zinc binding site. J Biol Chem, 268 (26), pp. 19292-19298. | Show Abstract

It has been proposed that the binding of Zn2+ to alpha-lactalbumin switches the conformation to one akin to a state intermediate in the folding of the protein. However, the high resolution x-ray crystal structure of human alpha-lactalbumin-Zn2+ complex at 1.7-A resolution (pH 7.6) does not reveal any significant change in conformation from the native state. The Zn2+ ion binds specifically in the "cleft" of alpha-lactalbumin (the region which forms the active site of the homologous protein lysozyme). This may suggest a possible role for Zn2+ binding in lactose synthase complex. The coordination of the Zn2+ ion involves a symmetry-related molecule in the crystal, the crystal contacts being stabilized by a SO4(2-) ion bound at the interface between three molecules.

PICKFORD M, GARMAN E, JONES E, STUART D. 1993. A DESIGN OF CRYSTAL MOUNTING CELL THAT ALLOWS THE CONTROLLED VARIATION OF HUMIDITY AT THE PROTEIN CRYSTAL DURING X-RAY-DIFFRACTION JOURNAL OF APPLIED CRYSTALLOGRAPHY, 26 (3), pp. 465-466. | Read more

Logan D, Abu-Ghazaleh R, Blakemore W, Curry S, Jackson T, King A, Lea S, Lewis R, Newman J, Parry N. 1993. Structure of a major immunogenic site on foot-and-mouth disease virus. Nature, 362 (6420), pp. 566-568. | Show Abstract | Read more

Attachment of foot-and-mouth disease virus (FMDV) to its cellular receptor involves a long and highly antigenic loop containing the conserved sequence, Arg-Gly-Asp, a motif known to be a recognition element in many integrin-dependent cell adhesion processes. In our original crystal structure of FMDV the Arg-Gly-Asp-containing loop ('the loop'), located between beta-strands G and H of capsid protein VP1, was disordered and hence essentially invisible. We previously surmised that its disorder is enhanced by a disulphide bond linking the base of the loop (Cys 134) to Cys 130 of VP2 (ref. 8). We report here the crystal structure of the virus in which this disulphide is reduced. Reduced virus retains infectivity and serological experiments suggest that some of the loop's internal structure is conserved. But here its structure has become sufficiently ordered to allow us to describe an unambiguous conformation, which we relate to some key biological properties of the virus.

Stammers DK, Delves C, Ballantine S, Jones EY, Stuart DI, Achari A, Bryant PK, Champness JN. 1993. Preliminary crystallographic data for Pneumocystis carinii dihydrofolate reductase. J Mol Biol, 230 (2), pp. 679-680. | Show Abstract | Read more

Dihydrofolate reductase from Pneumocystis carinii has been crystallized in a form suitable for high resolution X-ray diffraction studies. Recombinant enzyme that had been refolded following solubilization in guanidinium hydrochloride was crystallized as both a ternary complex with the cofactor NADPH and the inhibitor trimethoprim as well as a binary complex with NADPH. The two types of complex crystallized isomorphously from polyethylene glycol using sitting-drop vapour diffusion. The crystals were of space group P2(1) with unit cell parameters, a = 69.9 A, b = 43.6 A, c = 37.6 A, beta = 117.7 degrees, with one molecule per asymmetric unit. The crystals diffracted to 1.8 A resolution.

Davis SJ, Puklavec MJ, Ashford DA, Harlos K, Jones EY, Stuart DI, Williams AF. 1993. Expression of soluble recombinant glycoproteins with predefined glycosylation: application to the crystallization of the T-cell glycoprotein CD2. Protein Eng, 6 (2), pp. 229-232. | Read more

JONES E, STUART D, GARMAN E, GRIEST R, PHILLIPS D, TAYLOR G, MATSUMOTO O, DARBY G, LARDER B, LOWE D et al. 1993. THE GROWTH AND CHARACTERIZATION OF CRYSTALS OF HUMAN-IMMUNODEFICIENCY-VIRUS (HIV) REVERSE-TRANSCRIPTASE JOURNAL OF CRYSTAL GROWTH, 126 (2-3), pp. 261-269. | Show Abstract | Read more

Extensive studies on the crystallization of HIV-1 reverse transcriptase (RT) have yielded several crystal forms, two of which show diffraction to minimum Bragg spacings of 6 Å or better. Type 1 crystals belong to the space group P212121 with unit cell dimensions a = 147 A ̊, b = 190 A ̊ and c = 182 A ̊. Crystal density measurement indicate a very high crystal solvent content of 77% consistent with the presence of two RT heterodimers (66k/51k) per crystallographic asymmetric unit. These crystals are suitable for a low resolution determination of the apoenzyme structure. The second well ordered crystal form (space group P4222 with unit cell dimensions a = b = 120 A ̊, c = 320 A ̊) results from the co-crystallization of RT heterodimer and a double-stranded DNA oligonucleotide. Crystal density measurements again yield a relatively high value for the solvent content (7%; one RT heterodimer per crystallographic asymmetric unit) and elemental analysis indicates that one DNA oligonucleotide is associated with each RT heterodimer. This is consistent with each heterodimer possessing a single, competent, active site. © 1993.

Basak AK, Stuart DI, Roy P. 1992. Preliminary crystallographic study of bluetongue virus capsid protein, VP7. J Mol Biol, 228 (2), pp. 687-689. | Show Abstract | Read more

Bluetongue virus serotype 10 (BTV-10) VP7, expressed by insect cells infected with the recombinant baculovirus, has been purified and crystallized. Two crystal forms suitable for X-ray analysis have been obtained. Type I crystals belong to space group P6(3)22 with a = b = 95.2 A, c = 181.0 A, alpha = beta = 90 degrees gamma = 120.0 degrees, and contain a single subunit in the crystallographic asymmetric unit. They diffract to dmin = 3.0 A. Type II crystals belong to space group P2(1) with a = 69.4 A, b = 97.1 A, c = 71.4 A, beta = 109.0 degrees, and contain a trimer in the crystallographic asymmetric unit. They diffract to dmin = 2.1 A. These results, together with solution studies, show that the molecule is a trimer.

Jones EY, Davis SJ, Williams AF, Harlos K, Stuart DI. 1992. Crystal structure at 2.8 A resolution of a soluble form of the cell adhesion molecule CD2. Nature, 360 (6401), pp. 232-239. | Show Abstract | Read more

The crystal structure of a soluble form of the T lymphocyte antigen CD2 provides the first complete view of the extracellular region of a cell adhesion molecule. The topology of the molecule, which comprises two immunoglobulin-like domains, is the same as that of the first two domains of CD4 but the relative domain orientation is altered by a fairly flexible linker region. The putative ligand-binding beta-sheet forms a flat surface towards the top of the molecule. Crystal contacts between these surfaces suggest a plausible model for the adhesive interaction.

Jones EY, Stuart DI, Walker NP. 1992. Crystal structure of TNF. Immunol Ser, 56 pp. 93-127.

Jones EY, Walker NP, Stuart DI. 1991. Methodology employed for the structure determination of tumour necrosis factor, a case of high non-crystallographic symmetry. Acta Crystallogr A, 47 ( Pt 6) (6), pp. 753-770. | Show Abstract | Read more

The structure of the protein tumour necrosis factor (TNF) was determined from crystals of space group P3(1)21 which contain six copies of the TNF monomer per crystallographic asymmetric unit [Jones, Stuart & Walker (1989). Nature (London), 338, 225-228]. The nature of these crystals (relatively high crystallographic symmetry coupled with multiple copies of the protein in the asymmetric unit) led to some peculiarly challenging problems at several points in the structure determination. In particular, (1) self-rotation function calculations failed to yield clearly interpretable solutions, (2) the analysis of difference Patterson maps for heavy-atom derivatives required the development of a Patterson search program suite GROPAT. The redundancy in the asymmetric unit allowed refinement of poor-quality isomorphous phases at 4 A resolution and phase extension from 4 to 2.9 A resolution using real-space symmetry averaging and solvent flattening in the absence of any isomorphous phase information. Despite further difficulties caused by structural differences between the six independent copies of the monomer the resultant electron density map was of high quality and proved to be easily interpretable.

Acharya KR, Ren JS, Stuart DI, Phillips DC, Fenna RE. 1991. Crystal structure of human alpha-lactalbumin at 1.7 A resolution. J Mol Biol, 221 (2), pp. 571-581. | Show Abstract | Read more

The three-dimensional X-ray structure of human alpha-lactalbumin, an important component of milk, has been determined at 1.7 A (0.17 nm) resolution by the method of molecular replacement, using the refined structure of baboon alpha-lactalbumin as the model structure. The two proteins are known to have more than 90% amino acid sequence identity and crystallize in the same orthorhombic space group, P2(1)2(1)2. The crystallographic refinement of the structure using the simulated annealing method, resulted in a crystallographic R-factor of 0.209 for the 11,373 observed reflections (F greater than or equal to 2 sigma (F)) between 8 and 1.7 A resolution. The model comprises 983 protein atoms, 90 solvent atoms and a bound calcium ion. In the final model, the root-mean-square deviations from ideality are 0.013 A for covalent bond distances and 2.9 degrees for bond angles. Superposition of the human and baboon alpha-lactalbumin structures yields a root-mean-square difference of 0.67 A for the 123 structurally equivalent C alpha atoms. The C terminus is flexible in the human alpha-lactalbumin molecule. The striking structural resemblance between alpha-lactalbumins and C-type lysozymes emphasizes the homologous evolutionary relationship between these two classes of proteins.

Jones EY, Walker NP, Stuart DI. 1991. Methodology employed for the structure determination of tumour necrosis factor, a case of high non-crystallographic symmetry. Acta Crystallographica Section A: Foundations of Crystallography, 47 | Show Abstract | Read more

The structure of the protein tumour necrosis factor (TNF) was determined from crystals of space group P3(1)21 which contain six copies of the TNF monomer per crystallographic asymmetric unit [Jones, Stuart & Walker (1989). Nature (London), 338, 225-228]. The nature of these crystals (relatively high crystallographic symmetry coupled with multiple copies of the protein in the asymmetric unit) led to some peculiarly challenging problems at several points in the structure determination. In particular, (1) self-rotation function calculations failed to yield clearly interpretable solutions, (2) the analysis of difference Patterson maps for heavy-atom derivatives required the development of a Patterson search program suite GROPAT. The redundancy in the asymmetric unit allowed refinement of poor-quality isomorphous phases at 4 A resolution and phase extension from 4 to 2.9 A resolution using real-space symmetry averaging and solvent flattening in the absence of any isomorphous phase information. Despite further difficulties caused by structural differences between the six independent copies of the monomer the resultant electron density map was of high quality and proved to be easily interpretable.

Acharya KR, Stuart DI, Phillips DC, Scheraga HA. 1990. A critical evaluation of the predicted and X-ray structures of alpha-lactalbumin. J Protein Chem, 9 (5), pp. 549-563. | Show Abstract | Read more

The rapidly increasing availability of protein amino-acid sequences, many of which have been determined from the corresponding gene sequences, has intensified interest in the prediction of related protein structures when the three-dimensional structure of another member of the family is known. The study of bovine alpha-Lactalbumin provides a classic example in which the three-dimensional structure was predicted, first by Browne et al. (1969) and later by Warme et al. (1974), from the three-dimensional structure of hen-egg-white lysozyme (Blake et al., 1965), taking into account the striking relationship between the amino acid sequences of the two proteins. A comprehensive comparison of these models with the structure of baboon alpha-Lactalbumin derived from X-ray crystallography (Acharya et al., 1989) is presented. The models mostly compare well with the experimentally determined structure except in the flexible C-terminal region of the molecule (rms deviation on C alpha s of residues 1-95, 1.1 A).

Clark GR, Brown DG, Sanderson MR, Chwalinski T, Neidle S, Veal JM, Jones RL, Wilson WD, Zon G, Garman E. 1990. Crystal and solution structures of the oligonucleotide d(ATGCGCAT)2: a combined X-ray and NMR study. Nucleic Acids Res, 18 (18), pp. 5521-5528. | Show Abstract | Read more

A combined crystal-structure determination and NMR analysis of the octanucleotide d(ATGCGCAT)2 is reported. The X-ray analysis shows that the structure is A-form duplex in crystal state. The NMR study shows that in solution this sequence is B-type. The conformational results from each technique are presented in detail. The implications of these findings in terms of conformational flexibility and ligand binding are discussed.

Brown DG, Sanderson MR, Skelly JV, Jenkins TC, Brown T, Garman E, Stuart DI, Neidle S. 1990. Crystal structure of a berenil-dodecanucleotide complex: the role of water in sequence-specific ligand binding. EMBO J, 9 (4), pp. 1329-1334. | Show Abstract

The three-dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti-trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5'-AAT-3' region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen-bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen-bonded contact with an amidinium nitrogen atom, N3 of the 5' end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.

Jones EY, Stuart DI, Walker NP. 1990. The three-dimensional structure of tumour necrosis factor. Prog Clin Biol Res, 349 pp. 321-327.

Acharya KR, Stuart DI, Walker NP, Lewis M, Phillips DC. 1989. Refined structure of baboon alpha-lactalbumin at 1.7 A resolution. Comparison with C-type lysozyme. J Mol Biol, 208 (1), pp. 99-127. | Show Abstract | Read more

The solution of the structure of alpha-lactalbumin from baboon milk (Papio cynocephalus) at 4.5 A resolution using the isomorphous replacement method has been reported previously. Initial refinement on the basis of these low-resolution studies was not successful because of the poor isomorphism of the best heavy-atom derivative. Because of the striking similarity between the structure of lysozyme and alpha-lactalbumin, a more cautious molecular replacement approach was tried to refine the model. Using hen egg-white lysozyme as the starting model, preliminary refinement was performed using heavily constrained least-squares minimization in reciprocal space. The model was further refined using stereochemical restraints at 1.7 A resolution to a conventional crystallographic residual of 0.22 for 1141 protein atoms. In the final model, the root-mean-square deviation from ideality for bond distances is 0.015 A, and for angle distances it is 0.027 A. The refinement was carried out using the human alpha-lactalbumin sequence and "omit maps" calculated during the course of refinement indicated eight possible sequence changes in the baboon alpha-lactalbumin X-ray sequence. During the refinement, a tightly bound calcium ion and 150 water molecules, of which four are internal, have been located. Some of the water molecules were modelled for disordered side-chains. The co-ordination around the calcium is a slightly distorted pentagonal bipyramid. The Ca-O distances vary from 2.2 A to 2.6 A, representing a tight calcium-binding loop in the structure. The calcium-binding fold only superficially resembles the "EF-hand" and presumably has no evolutionary relationship with other EF-hand structures. The overall structure of alpha-lactalbumin is very similar to that of lysozyme. All large deviations occur in the loops where all sequence deletions and insertions are found. The C terminus appears to be rather flexible in alpha-lactalbumin compared to lysozyme. The experimental evidence supports the earlier predictions for the alpha-lactalbumin structure that were based upon the assumption that alpha-lactalbumin and lysozyme have similar three-dimensional structures, with minimal deletions and insertions. A detailed comparison of the two structures shows striking features as well as throwing some light on the evolution of these two proteins from a common precursor.

Oikonomakos NG, Acharya KR, Melpidou AE, Stuart DI, Johnson LN. 1989. The binding of beta-glycerophosphate to glycogen phosphorylase b in the crystal. Arch Biochem Biophys, 270 (1), pp. 62-68. | Show Abstract | Read more

The binding of beta-glycerophosphate (glycerol-2-P) to glycogen phosphorylase b in the crystal has been studied by X-ray diffraction at 3 A resolution. Glycerol-2-P binds to the allosteric effector site in a position close to that of AMP, glucose-6-P, UDP-Glc, and phosphate. In this position, glycerol-2-P is stabilized through interactions of its phosphate moiety with the guanidinium groups of Arg 309 and Arg 310 which undergo conformational changes, and the hydroxyl group of Tyr 75, while the same residues and solvent are involved in van der Waals interactions with the remaining part of the molecule. Kinetic experiments indicate that glycerol-2-P partially competes with both the activator (AMP) and the inhibitor (glucose 6-phosphate) of phosphorylase b. A comparison of the positions of glycerol-2-P, AMP, glucose 6-phosphate, UDP-Glc, and Pi at the allosteric site is presented.

Jones EY, Stuart DI, Walker NP. 1989. Structure of tumour necrosis factor. Nature, 338 (6212), pp. 225-228. | Show Abstract | Read more

Tumour necrosis factor is a trimeric molecule, each subunit of which consists of an antiparallel beta-sandwich. Individual subunits from the trimer by a novel edge-to-face packing of beta-sheets. A comparison of the subunit fold with that of other proteins reveals a remarkable similarity to the 'jelly-roll' structural motif characteristic of viral coat proteins.

Skelly JV, Suter DA, Knox RJ, Garman E, Stuart DI, Sanderson MR, Roberts JJ, Neidle S. 1989. Preliminary crystallographic data for NAD(P)H quinone reductase isolated from the Walker 256 rat carcinoma cell line. J Mol Biol, 205 (3), pp. 623-624. | Show Abstract | Read more

An NAD(P)H quinone reductase isolated from Walker rat 256 carcinoma cells has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell parameters a = 168.15 A, b = 105.09 A and c = 67.38 A and contain four monomeric or two dimeric enzyme molecules per asymmetric unit. Diffraction extends beyond 2.3 A resolution.

Sprang SR, Acharya KR, Goldsmith EJ, Stuart DI, Varvill K, Fletterick RJ, Madsen NB, Johnson LN. 1988. Structural changes in glycogen phosphorylase induced by phosphorylation. Nature, 336 (6196), pp. 215-221. | Show Abstract | Read more

A comparison of the refined crystal structures of dimeric glycogen phosphorylase b and a reveals structural changes that represent the first step in the activation of the enzyme. On phosphorylation of serine-14, the N-terminus of each subunit assumes an ordered helical conformation and binds to the surface of the dimer. The consequent structural changes at the N- and C-terminal regions lead to strengthened interactions between subunits and alter the binding sites for allosteric effectors and substrates.

Oikonomakos NG, Acharya KR, Stuart DI, Melpidou AE, McLaughlin PJ, Johnson LN. 1988. Uridine(5')diphospho(1)-alpha-D-glucose. A binding study to glycogen phosphorylase b in the crystal. Eur J Biochem, 173 (3), pp. 569-578. | Show Abstract | Read more

UDP-glucose is an R-state inhibitor of glycogen phosphorylase b, competitive with the substrate, glucose 1-phosphate and noncompetitive with the allosteric activator, AMP. Diffusion of 100 mM UDP-glucose into crystals of phosphorylase b resulted in a difference Fourier synthesis at 0.3-nm resolution that showed two peaks: (a) binding at the allosteric site and (b) binding at the catalytic site. At the allosteric site the whole of the UDP-glucose molecule can be located. It is in a well defined folded conformation with its uracil portion in a similar position to that observed for the adenine of AMP. The uracil and the glucose moieties stack against the aromatic side chains of Tyr-75 and Phe-196, respectively. The phosphates of the pyrophosphate component interact with Arg-242, Arg-309 and Arg-310. At the catalytic site, the glucose-1-P component of UDP-glucose is firmly bound in a position similar to that observed for glucose 1-phosphate. The pyrophosphate is also well located with the glucose phosphate interacting with the main-chain NH groups at the start of the glycine-loop alpha helix and the uridine phosphate interacting through a water molecule with the 5'-phosphate of the cofactor pyridoxal phosphate and with the side chains of residues Tyr-573, Lys-574 and probably Arg-569. However the position of the uridine cannot be located although analysis by thin-layer chromatography showed that no degradation had taken place. Binding of UDP-glucose to the catalytic site promotes extensive conformational changes. The loop 279-288 which links the catalytic site to the nucleoside inhibitor site is displaced and becomes mobile. Concomitant movements of residues His-571, Arg-569, and the loop 378-383, together with the major loop displacement, result in an open channel to the catalytic site. Comparison with other structural results shows that these changes form an essential feature of the T to R transition. They allow formation of the phosphate recognition site at the catalytic site and destroy the nucleoside inhibitor site. Kinetic experiments demonstrate that UDP-glucose activates the enzyme in the presence of high concentrations of the weak activator IMP, because of its ability to decrease the affinity of IMP for the inhibitor site.

Hajdu J, Acharya KR, Stuart DI, Barford D, Johnson LN. 1988. Catalysis in enzyme crystals. Trends Biochem Sci, 13 (3), pp. 104-109. | Show Abstract | Read more

New developments in X-ray crystallography may produce three-dimensional movies of catalysis and structural changes. © 1988.

Oikonomakos NG, Johnson LN, Acharya KR, Stuart DI, Barford D, Hajdu J, Varvill KM, Melpidou AE, Papageorgiou T, Graves DJ. 1987. Pyridoxal phosphate site in glycogen phosphorylase b: structure in native enzyme and in three derivatives with modified cofactors. Biochemistry, 26 (25), pp. 8381-8389. | Show Abstract | Read more

The detailed environment of the essential cofactor pyridoxal 5'-phosphate in glycogen phosphorylase b, resulting from crystallographic refinement at 1.9-A resolution, is described. The pyridoxal ring is buried in a nonpolar site containing three aromatic rings while the 5'-phosphate group is highly solvated and makes only three direct contacts to the protein. The pyridine nitrogen interacts via a water with protein atoms [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. The crystal structures of three active derivatives of phosphorylase reconstituted with 5'-deoxypyridoxal 5'-methylenephosphonate (PDMP), 6-fluoropyridoxal 5'-phosphate (6-FPLP), and pyridoxal (PL) in place of the natural cofactor have been determined at 2.5-A resolution. The results for PDMP-phosphorylase show a closer proximity of the phosphonate group to the NZ atom of a lysine (Lys-574) than that observed in the native enzyme, consistent with 31P NMR studies that have shown a change in ionization state of the phosphonate group compared to the native cofactor phosphate. The replacement of the polar 5'-ester linkage by a CH2 group results in a small shift of a water and its hydrogen-bonded tyrosine (Tyr-648). In 6-FPLP-phosphorylase the fluorine is accommodated with no significant change in structure. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1.(ABSTRACT TRUNCATED AT 250 WORDS)

Johnson LN, Acharya KR, Stuart DI, Barford D, Oikonomakos NG, Hajdu J, Varvill KM. 1987. Phosphate-recognition sites in catalysis and control of glycogen phosphorylase b. Biochem Soc Trans, 15 (5), pp. 1001-1005. | Read more

Phillips DC, Acharya KR, Handoll HH, Stuart DI. 1987. From lysozyme to alpha-lactalbumin: protein engineering and evolution. Biochem Soc Trans, 15 (4), pp. 737-744. | Read more

Smith SG, Lewis M, Aschaffenburg R, Fenna RE, Wilson IA, Sundaralingam M, Stuart DI, Phillips DC. 1987. Crystallographic analysis of the three-dimensional structure of baboon alpha-lactalbumin at low resolution. Homology with lysozyme. Biochem J, 242 (2), pp. 353-360. | Show Abstract | Read more

The crystal structure of baboon alpha-lactalbumin has been determined at 6 A and at 4.5 A (0.6 nm and 0.45 nm) resolution by the method of isomorphous replacement. The principal derivative was prepared by reducing a disulphide bridge in the crystals and inserting a mercury atom. Detailed comparison of the electron-density maps with corresponding maps of hen egg-white lysozyme shows that they are closely similar, with correlation coefficients of 0.57 and 0.44 at 6 A and 4.5 A resolution respectively. This result, in accordance with earlier predictions based upon comparisons of amino-acid sequences, provides further evidence that class C lysozymes and alpha-lactalbumins are homologous proteins and it is in keeping with the hypothesis that the alpha-lactalbumins evolved from a lysozyme precursor.

Hajdu J, Acharya KR, Stuart DI, McLaughlin PJ, Barford D, Oikonomakos NG, Klein H, Johnson LN. 1987. Catalysis in the crystal: synchrotron radiation studies with glycogen phosphorylase b. EMBO J, 6 (2), pp. 539-546. | Show Abstract

Direct observation of the progress of a catalysed reaction in crystals of glycogen phosphorylase b has been made possible through fast crystallographic data collection achieved at the Synchrotron Radiation source at Daresbury, UK. In the best experiments, data to 2.7 A resolution (some 108,300 measurements; 21,200 unique reflections) were measured in 25 min. In a series of time-resolved studies in which the control properties of the enzyme were exploited in order to slow down the reaction, the conversion of heptenitol to heptulose-2-phosphate, the phosphorylysis of maltoheptaose to yield glucose-1-phosphate and the oligosaccharide synthesis reaction involving maltotriose and glucose-1-phosphate have been monitored in the crystal. Changes in electron density in the difference Fourier maps are observed as the reaction proceeds not only at the catalytic site but also the allosteric and glycogen storage sites. Phosphorylase b is present in the crystals in the T state and under these conditions exhibits low affinity for both phosphate and oligosaccharide substrates. There are pronounced conformational changes associated with the formation and binding of the high-affinity dead-end product, heptulose-2-phosphate, which show that movement of an arginine residue, Arg 569, is critical for formation of the substrate-phosphate recognition site. The results are discussed with reference to proposals for the enzymic mechanism of phosphorylase. The feasibility for time-resolved studies on other systems and recent advances in this area utilizing Laue diffraction are also discussed.

Stuart DI, Acharya KR, Walker NP, Smith SG, Lewis M, Phillips DC. 1986. Alpha-lactalbumin possesses a novel calcium binding loop. Nature, 324 (6092), pp. 84-87. | Show Abstract | Read more

Calcium performs a unique role in biology, achieving biological effects through highly specific interactions with and modulation of target proteins. It has been proposed that calcium-modulated proteins possess a characteristic, evolutionarily related, binding fold, known as the EF-hand. The high-resolution X-ray structure of alpha-lactalbumin reveals a Ca2+ binding fold that resembles an EF-hand only superficially and presumably has no evolutionary relationship with it. However, there is clear homology with the corresponding loop in c-type lysozyme (the 'parent' molecule of alpha-lactalbumin). This study, at 1.7 A resolution, represents one of the most accurate analyses of a calcium binding protein yet reported.

Hajdu J, Acharya KR, Stuart DI, McLaughlin PJ, Barford D, Klein H, Johnson LN. 1986. Time-resolved structural studies on catalysis in the crystal with glycogen phosphorylase b. Biochem Soc Trans, 14 (3), pp. 538-541. | Read more

SANSOM M, STUART D, ACHARYA K, HAJDU J, MCLAUGHLIN P, JOHNSON L. 1985. GLYCOGEN PHOSPHORYLASE-B - THE MOLECULAR ANATOMY OF A LARGE REGULATORY ENZYME THEOCHEM-JOURNAL OF MOLECULAR STRUCTURE, 24 (1-2), pp. 3-25.

Stuart DI, Phillips DC. 1985. On the derivation of dynamic information from diffraction data. Methods Enzymol, 115 pp. 117-142.

Lorek A, Wilson KS, Sansom MS, Stuart DI, Stura EA, Jenkins JA, Zanotti G, Hajdu J, Johnson LN. 1984. Allosteric interactions of glycogen phosphorylase b. A crystallographic study of glucose 6-phosphate and inorganic phosphate binding to di-imidate-cross-linked phosphorylase b. Biochem J, 218 (1), pp. 45-60. | Show Abstract | Read more

The binding to glycogen phosphorylase b of glucose 6-phosphate and inorganic phosphate (respectively allosteric inhibitor and substrate/activator of the enzyme) were studied in the crystal at 0.3 nm (3A) resolution. Glucose 6-phosphate binds in the alpha-configuration at a site that is close to the AMP allosteric effector site at the subunit-subunit interface and promotes several conformational changes. The phosphate-binding site of the enzyme for glucose 6-phosphate involves contacts to two cationic residues, Arg-309 and Lys-247. This site is also occupied in the inorganic-phosphate-binding studies and is therefore identified as a high-affinity phosphate-binding site. It is distinct from the weaker phosphate-binding site of the enzyme for AMP, which is 0.27 nm (2.7A) away. The glucose moiety of glucose 6-phosphate and the adenosine moiety of AMP do not overlap. The results provide a structural explanation for the kinetic observations that glucose 6-phosphate inhibition of AMP activation of phosphorylase b is partially competitive and highly co-operative. The results suggest that the transmission of allosteric conformational changes involves an increase in affinity at phosphate-binding sites and relative movements of alpha-helices. In order to study glucose 6-phosphate and phosphate binding it was necessary to cross-link the crystals. The use of dimethyl malondi-imidate as a new cross-linking reagent in protein crystallography is discussed.

McLaughlin PJ, Stuart DI, Klein HW, Oikonomakos NG, Johnson LN. 1984. Substrate-cofactor interactions for glycogen phosphorylase b: a binding study in the crystal with heptenitol and heptulose 2-phosphate. Biochemistry, 23 (24), pp. 5862-5873. | Show Abstract | Read more

The structural relationships between substrate and pyridoxal phosphate in glycogen phosphorylase b (EC 2.4.1.1) have been studied by X-ray diffraction experiments at 3-A resolution. Recent work [Klein, H. W., Im, M. J., & Helmreich, E. J. M. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, A. E., Ed.) pp 147-160, Liss, New York] has shown that phosphorylase in the presence of inorganic phosphate catalyzes the conversion of heptenitol to heptulose 2-phosphate. The latter compound is a dead-end product and a most potent inhibitor (Ki = 14 microM). The X-ray diffraction studies show that heptenitol binds at the catalytic site of phosphorylase in a position essentially identical with that observed for the glucopyranose moiety of glucose 1-phosphate. Incubation of a phosphorylase b crystal for 50 h in a solution containing the substrates heptenitol and inorganic phosphate and the activators AMP and maltohetaose resulted in the formation of a phosphorylated product bound at the active site. The structure of this product, as analyzed by a difference Fourier synthesis at 3 A, is consistent with that of heptulose 2-phosphate. Analysis of the surrounding soak solution by thin-layer chromatography showed that heptulose 2-phosphate was produced under these conditions. Heptulose 2-phosphate binds with its glucopyranose moiety in the same position as that for glucose 1-phosphate, but there is a marked difference in phosphate positions. The presence of the methyl group in the beta-configuration in heptulose 2-phosphate forces a change in the torsion angle O5-C1-O1-P from 117 degrees as observe in glucose 1-phosphate to -136 degrees in heptulose 2-phosphate. The "down" position of the phosphate (with respect to the crystallographic z axis) results in a change in the distance between the 5'-phosphorus atom of the pyridoxal phosphate and the phosphorus atom of the substrate from 6.8 (with glucose 1-phosphate) to 4.5 A (with heptulose 2-phosphate). The closest distance between the phosphate oxygen of the cofactor and a phosphate oxygen of heptulose 2-phosphate is 2.7 A, and it is assumed that there must be a hydrogen bond between them. These observations are consistent with the NMR experiments reported in the preceding paper in which sharing of a proton between heptulose 2-phosphate and pyridoxal 5'-phosphate is observed [Klein, H.W., Im, M. J., Palm, D., & Helmreich, E. J. M. (1984) Biochemistry (preceding paper in this issue)].(ABSTRACT TRUNCATED AT 400 WORDS)

Stura EA, Zanotti G, Babu YS, Sansom MS, Stuart DI, Wilson KS, Johnson LN, Van de Werve G. 1983. Comparison of AMP and NADH binding to glycogen phosphorylase b. J Mol Biol, 170 (2), pp. 529-565. | Show Abstract | Read more

The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.

Cited:

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WOS

WILSON K, STURA E, WILD D, TODD R, STUART D, BABU Y, JENKINS J, STANDING T, JOHNSON L, FOURME R et al. 1983. MACROMOLECULAR CRYSTALLOGRAPHY WITH SYNCHROTRON RADIATION .2. RESULTS JOURNAL OF APPLIED CRYSTALLOGRAPHY, 16 (FEB), pp. 28-41. | Read more

Jenkins JA, Johnson LN, Stuart DI, Stura EA, Wilson KS, Zanotti G. 1981. Phosphorylase: control and activity. Philos Trans R Soc Lond B Biol Sci, 293 (1063), pp. 23-41. | Show Abstract | Read more

Recent results from the crystallographic studies on glycogen phosphorylase b at 2 A resolution are reviewed with special reference to other themes of the meeting. The structural similarity of the fold of 150 residues in phosphorylase to the observed in lactate dehydrogenase is discussed and the binding sites for NADH in phosphorylase are described. The binding of the potent inhibitor glucose-1,2-cyclic phosphate to phosphorylase b in the crystal has been studied at 3 A resolution. The results are compared with those previously obtained for glucose-1-phosphate and discussed with reference to proposals for a mechanism of catalysis that involves the essential cofactor pyridoxal phosphate.

Stuart DI, Levine M, Muirhead H, Stammers DK. 1979. Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 A. J Mol Biol, 134 (1), pp. 109-142. | Show Abstract | Read more

The structure of pyruvate kinase (EC 2.7.1.40) has been determined from a 2.6 Å resolution electron density map. This map shows more detail than the previous 3.1 Å map (Stammers & Muirhead, 1977) and has enabled a detailed chain folding to be established for two out of the three domains which make up each of the four identical subunits. A provisional chain folding has been established for the third domain. The results have been briefly reported in a previous paper (Levine et al., 1978). Details of the structure determination and a further discussion of the results are presented in this paper. Domain A (the three domains of pyruvate kinase are referred to as A, B and C) can be described in terms of a cylindrical eight-stranded parallel β sheet and an outer coaxial cylinder of eight α helices. The α helices connect adjacent strands of the β sheet. Domain B is made up of a closed anti-parallel β sheet structure. Domain C is a five-stranded β sheet of which the fourth strand is anti-parallel and the rest parallel. These strands are also interconnected by α helices. Domain A can be dissected into eight consecutive β strand-α helix units starting from the N-terminus. The arrangement of these relative to each other can be most simply described by relating them to eight planes, each at 40 ° to the cylinder axis and symmetrically placed around the cylinder. When unit 2 is aligned with one of these planes then units 1, 3, 4, 5 and 8 are also closely aligned with a plane. This analysis is also applied to triosephosphate isomerase and a strikingly similar arrangement is found. A detailed comparison of the two structures is presented. Although the lack of a chemical sequence makes it difficult to identify the amino acid residues of pyruvate kinase, side-chains are clearly visible in the map and this information is correlated with the results of previous 6 Å substrate soaking experiments and with the structure of triosephosphate isomerase. The similarities and differences are discussed in terms of similarities and differences in the reactions catalysed and also of different subunit packing. © 1979.

Levine M, Muirhead H, Stammers DK, Stuart DI. 1978. Structure of pyruvate kinase and similarities with other enzymes: possible implications for protein taxonomy and evolution. Nature, 271 (5646), pp. 626-630. | Show Abstract | Read more

The structure determination of pyruvate kinase shows that each subunit of the tetrameric molecule consists of three domains. The largest of these domains has a remarkable similarity to the structure of triosephosphate isomerase. Another domain shows similarities to many other nucleotide binding proteins. We discuss these similarities and their implications for current arguments on protein taxonomy and evolution.

Stammers DK, Levine M, Stuart DI, Muirhead H. 1977. Structure of cat muscle pyruvate kinase at 0.26 nm resolution. Biochem Soc Trans, 5 (3), pp. 654-657. | Read more

Zhao Y, Ren J, Harlos K, Jones DM, Zeltina A, Bowden TA, Padilla-Parra S, Fry EE, Stuart DI. 2016. Toremifene interacts with and destabilizes the Ebola virus glycoprotein. Nature, 535 (7610), pp. 169-172. | Show Abstract | Read more

Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein–drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.

Zhu L, Wang X, Ren J, Kotecha A, Walter TS, Yuan S, Yamashita T, Tuthill TJ, Fry EE, Rao Z, Stuart DI. 2016. Structure of human Aichi virus and implications for receptor binding. Nat Microbiol, 1 (11), pp. 16150. | Show Abstract | Read more

Aichi virus (AiV), an unusual and poorly characterized picornavirus, classified in the genus Kobuvirus, can cause severe gastroenteritis and deaths in children below the age of five years, especially in developing countries(1,2). The seroprevalence of AiV is approximately 60% in children under the age of ten years and reaches 90% later in life(3,4). There is no available vaccine or effective antiviral treatment. Here, we describe the structure of AiV at 3.7 Å. This first high-resolution structure for a kobuvirus is intermediate between those of the enteroviruses and cardioviruses, with a shallow, narrow depression bounded by the prominent VP0 CD loops (linking the C and D strands of the β-barrel), replacing the depression known as the canyon, frequently the site of receptor attachment in enteroviruses. VP0 is not cleaved to form VP2 and VP4, so the 'VP2' β-barrel structure is complemented with a unique extended structure on the inside of the capsid. On the outer surface, a polyproline helix structure, not seen previously in picornaviruses is present at the C terminus of VP1, a position where integrin binding motifs are found in some other picornaviruses. A peptide corresponding to this polyproline motif somewhat attenuates virus infectivity, presumably blocking host-cell attachment. This may guide cellular receptor identification.

Hengrung N, El Omari K, Serna Martin I, Vreede FT, Cusack S, Rambo RP, Vonrhein C, Bricogne G, Stuart DI, Grimes JM, Fodor E. 2015. Crystal structure of the RNA-dependent RNA polymerase from influenza C virus. Nature, 527 (7576), pp. 114-117. | Show Abstract | Read more

Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome. In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching. Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5' and 3' termini of viral genome segments), showing FluPol in transcription pre-initiation states. Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new 'closed' conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols. The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication.

Ilca SL, Kotecha A, Sun X, Poranen MM, Stuart DI, Huiskonen JT. 2015. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes. Nat Commun, 6 pp. 8843. | Show Abstract | Read more

Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems.

Zhu L, Wang X, Ren J, Porta C, Wenham H, Ekström JO, Panjwani A, Knowles NJ, Kotecha A, Siebert CA et al. 2015. Structure of Ljungan virus provides insight into genome packaging of this picornavirus. Nat Commun, 6 pp. 8316. | Show Abstract | Read more

Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses.

Ji X, Axford D, Owen R, Evans G, Ginn HM, Sutton G, Stuart DI. 2015. Polyhedra structures and the evolution of the insect viruses. J Struct Biol, 192 (1), pp. 88-99. | Show Abstract | Read more

Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽ 1.5 Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These structures illustrate the effect of 400 million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule's evolution so that surface residues are almost as conserved as the internal residues.

Ren J, Wang X, Zhu L, Hu Z, Gao Q, Yang P, Li X, Wang J, Shen X, Fry EE et al. 2015. Structures of Coxsackievirus A16 Capsids with Native Antigenicity: Implications for Particle Expansion, Receptor Binding, and Immunogenicity. J Virol, 89 (20), pp. 10500-10511. | Show Abstract | Read more

UNLABELLED: Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the primary causes of the epidemics of hand-foot-and-mouth disease (HFMD) that affect more than a million children in China each year and lead to hundreds of deaths. Although there has been progress with vaccines for EV71, the development of a CVA16 vaccine has proved more challenging, and the EV71 vaccine does not give useful cross-protection, despite the capsid proteins of the two viruses sharing about 80% sequence identity. The structural details of the expanded forms of the capsids, which possess nonnative antigenicity, are now well understood, but high resolution information for the native antigenic form of CVA16 has been missing. Here, we remedy this with high resolution X-ray structures of both mature and natural empty CVA16 particles and also of empty recombinant viruslike particles of CVA16 produced in insect cells, a potential vaccine antigen. All three structures are unexpanded native particles and antigenically identical. The recombinant particles have recruited a lipid moiety to stabilize the native antigenic state that is different from the one used in a natural virus infection. As expected, the mature CVA16 virus is similar to EV71; however, structural and immunogenic comparisons highlight differences that may have implications for vaccine production. IMPORTANCE: Hand-foot-and-mouth disease is a serious public health threat to children in Asian-Pacific countries, resulting in millions of cases. EV71 and CVA16 are the two dominant causative agents of the disease that, while usually mild, can cause severe neurological complications, leading to hundreds of deaths. EV71 vaccines do not provide protection against CVA16. A CVA16 vaccine or bivalent EV71/CVA16 vaccine is therefore urgently needed. We report atomic structures for the mature CVA16 virus, a natural empty particle, and a recombinant CVA16 virus-like particle that does not contain the viral genome. All three particles have similar structures and identical antigenicity. The recombinant particles, produced in insect cells (a system suitable for making vaccine antigen), are stabilized by recruiting from the insect cells a small molecule that is different from that used by the virus in a normal infection. We present structural and immunogenic comparisons with EV71 to facilitate structure-based drug design and vaccine development.

Kotecha A, Seago J, Scott K, Burman A, Loureiro S, Ren J, Porta C, Ginn HM, Jackson T, Perez-Martin E et al. 2015. Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design. Nat Struct Mol Biol, 22 (10), pp. 788-794. | Show Abstract | Read more

Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

Ren J, Wang X, Hu Z, Gao Q, Sun Y, Li X, Porta C, Walter TS, Gilbert RJ, Zhao Y et al. 2013. Picornavirus uncoating intermediate captured in atomic detail. Nat Commun, 4 pp. 1929. | Show Abstract | Read more

It remains largely mysterious how the genomes of non-enveloped eukaryotic viruses are transferred across a membrane into the host cell. Picornaviruses are simple models for such viruses, and initiate this uncoating process through particle expansion, which reveals channels through which internal capsid proteins and the viral genome presumably exit the particle, although this has not been clearly seen until now. Here we present the atomic structure of an uncoating intermediate for the major human picornavirus pathogen CAV16, which reveals VP1 partly extruded from the capsid, poised to embed in the host membrane. Together with previous low-resolution results, we are able to propose a detailed hypothesis for the ordered egress of the internal proteins, using two distinct sets of channels through the capsid, and suggest a structural link to the condensed RNA within the particle, which may be involved in triggering RNA release.

Porta C, Kotecha A, Burman A, Jackson T, Ren J, Loureiro S, Jones IM, Fry EE, Stuart DI, Charleston B. 2013. Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen. PLoS Pathog, 9 (3), pp. e1003255. | Show Abstract | Read more

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.

El Omari K, Iourin O, Harlos K, Grimes JM, Stuart DI. 2013. Structure of a pestivirus envelope glycoprotein E2 clarifies its role in cell entry. Cell Rep, 3 (1), pp. 30-35. | Show Abstract | Read more

Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.

Wang X, Peng W, Ren J, Hu Z, Xu J, Lou Z, Li X, Yin W, Shen X, Porta C et al. 2012. A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat Struct Mol Biol, 19 (4), pp. 424-429. | Show Abstract | Read more

Enterovirus 71 (EV71) is a major agent of hand, foot and mouth disease in children that can cause severe central nervous system disease and death. No vaccine or antiviral therapy is available. High-resolution structural analysis of the mature virus and natural empty particles shows that the mature virus is structurally similar to other enteroviruses. In contrast, the empty particles are markedly expanded and resemble elusive enterovirus-uncoating intermediates not previously characterized in atomic detail. Hydrophobic pockets in the EV71 capsid are collapsed in this expanded particle, providing a detailed explanation of the mechanism for receptor-binding triggered virus uncoating. These structures provide a model for enterovirus uncoating in which the VP1 GH loop acts as an adaptor-sensor for cellular receptor attachment, converting heterologous inputs to a generic uncoating mechanism, highlighting new opportunities for therapeutic intervention.

Mapping RNA virus replication factories in molecular detail

Technology advances in electron imaging and sample preparation mean that is now possible not only to generate high resolution structures of isolated virus particles (for example Zhu et al, 2016) but also to begin to map the key events in the viral life cycle within the cell at molecular resolution.The virus systems selected for this are representatives from two families of RNA virus that we have studied for some years, the Picornaviridae and the Reoviridae. From these families which include a ...

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Structural biology of cell surface receptors.

Many major questions in receptor biology centre on how interactions occurring outside the cell trigger signalling inside the cell. To answer such questions for the biomedically relevant systems used in human cells we need to be able to express, purify and structurally analyse membrane spanning glycoproteins. Many of the necessary methodologies have only very recently been developed, and these are now bearing fruit, so this is a very exciting time for the field of receptor research. We are one ...

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